Methods for treating muscle diseases and disorders

ABSTRACT

The invention relates to methods of treating diseases and disorders of the muscle tissues in a vertebrate by the administration of compounds which bind the p185 erbB2  receptor. These compounds are found to cause increased differentiation and survival of cardiac, skeletal and smooth muscle.

This is a divisional of copending application U.S. Ser. No. 08/209,204,filed Mar. 8, 1994, which is a continuation in part of application U.S.Ser. No. 08/059,022 filed May 6, 1993 (abandoned). This is also acontinuation-in-part of application U.S. Ser. No. 08/036,555, filed Mar.24, 1993, now U.S. Pat. No. 5,530,109, which is a continuation-in-partof application U.S. Ser. No. 07/965,173, filed Oct. 23, 1992(abandoned), application U.S. Ser. No. 07/940,389, filed Sep. 3, 1992(abandoned), application U.S. Ser. No. 07/907,138, filed Jun. 30, 1992(abandoned), and application U.S. Ser. No. 07/863,703, filed Apr. 3,1992 (abandoned).

BACKGROUND OF THE INVENTION

The invention relates to prophylactic or affirmative treatment ofdiseases and disorders of the musculature by administering polypeptidesfound in vertebrate species, which polypeptides are growth,differentiation and survival factors for muscle cells.

Muscle tissue in adult vertebrates will regenerate from reservemyoblasts called satellite cells. Satellite cells are distributedthroughout muscle tissue and are mitotically quiescent in the absence ofinjury or disease. Following muscle injury or during recovery fromdisease, satellite cells will reenter the cell cycle, proliferate and 1)enter existing muscle fibers or 2) undergo differentiation intomultinucleate myotubes which form new muscle fiber. The myoblastsultimately yield replacement muscle fibers or fuse into existing musclefibers, thereby increasing fiber girth by the synthesis of contractileapparatus components. This process is illustrated, for example, by thenearly complete regeneration which occurs in mammals following inducedmuscle fiber degeneration; the muscle progenitor cells proliferate andfuse together regenerating muscle fibers.

Several growth factors which regulate the proliferation anddifferentiation of adult (and embryonic) myoblasts in vitro have beenidentified. Fibroblast growth factor (FGF) is mitogenic for muscle cellsand is an inhibitor of muscle differentiation. Transforming growthfactor β (TGFβ) has no effect on myoblast proliferation, but is aninhibitor of muscle differentiation. Insulin-like growth factors (IGFs)have been shown to stimulate both myoblast proliferation anddifferentiation in rodents. Platelet derived growth factor (PDGF) isalso mitogenic for myoblasts and is a potent inhibitor of muscle celldifferentiation. (For a review of myoblast division and differentiationsee: Florini and Magri, 1989:256:C701–C711).

In vertebrate species both muscle tissue and neurons are potentialsources of factors which stimulate myoblast proliferation anddifferentiation. In diseases affecting the neuromuscular system whichare neural in origin (i.e., neurogenic), the muscle tissue innervated bythe affected nerve becomes paralyzed and wastes progressively. Duringperipheral nerve regeneration and recovery from neurologic and myopathicdisease, neurons may provide a source of growth factors which elicit themuscle regeneration described above and provide a mechanism for musclerecovery from wasting and atrophy.

A recently described family of growth factors, the neuregulins, aresynthesized by motor neurons (Marchioni et al. Nature 362:313, 1993) andinflammatory cells (Tarakhovsky et al., Oncogene 6:2187–2196 (1991)).The neuregulins and related p185^(erbB2) binding factors have beenpurified, cloned and expressed (Benveniste et al., PNAS 82:3930–3934,1985; Kimura et al., Nature 348:257–260, 1990; Davis and Stroobant, J.Cell. Biol. 110:1353–1360, 1990; Wen et al., Cell 69:559, 1992; Yardenand Ullrich, Ann. Rev. Biochem. 57:443, 1988; Holmes et al., Science256:1205, 1992; Dobashi et al., Proc. Natl. Acad. Sci. 88:8582, 1991;Lupu et al., Proc. Natl. Acad. Sci. 89:2287, 1992). Recombinantneuregulins have been shown to be mitogenic for peripheral glia(Marchionni et al., Nature 362:313, 1993) and have been shown toinfluence the formation of the neuromuscular junction (Falls et al.,Cell 72:801, 1993). Thus the regenerating neuron and the inflammatorycells associated with the recovery from neurogenic disease and nerveinjury provide a source of factors which coordinate the remyelination ofmotor neurons and their ability to form the appropriate connection withtheir target. After muscle has been reinnervated the motor neuron mayprovide factors to muscle, stimulating muscle growth and survival.

Currently, there is no useful therapy for the promotion of muscledifferentiation and survival. Such a therapy would be useful fortreatment of a variety of neural and muscular diseases and disorders.

SUMMARY OF THE INVENTION

We have discovered that increased mitogenesis differentiation andsurvival of muscle cells may be achieved using proteins heretoforedescribed as glial growth factors, acetylcholine receptor inducingactivity (ARIA), heregulins, neu differentiation factor, and, moregenerally, neuregulins. We have discovered that these compounds arecapable of inducing both the proliferation of muscle cells and thedifferentiation and survival of myotubes. These phenomena may occur incardiac and smooth muscle tissues in addition to skeletal muscletissues. Thus, the above compounds, regulatory compounds which inducesynthesis of these compounds, and small molecules which mimic thesecompounds by binding to the receptors on muscle or by stimulatingthrough other means the second messenger systems activated by theligand-receptor complex are all extremely useful as prophylactic andaffirmative therapies for muscle diseases.

A novel aspect of the invention involves the use of the above namedproteins as growth factors to induce the mitogenesis, survival, growthand differentiation of muscle cells. Treating of the muscle cells toachieve these effects may be achieved by contacting muscle cells with apolypeptide described herein. The treatments may be provided to slow orhalt net muscle loss or to increase the amount or quality of musclepresent in the vertebrate.

These factors may be used to produce muscle cell mitogenesis,differentiation, and survival in a vertebrate (preferably a mammal, morepreferably a human) by administering to the vertebrate an effectiveamount of a polypeptide or a related compound. Neuregulin effects onmuscle may occur, for example, by causing an increase in muscleperformance by inducing the synthesis of particular isoforms of thecontractile apparatus such as the myosin heavy chain slow and fastisoforms; by promoting muscle fiber survival via the induction ofsynthesis of protective molecules such as, but not limited to,dystrophin; and/or by increasing muscle innervation by, for example,increasing acetylcholine receptor molecules at the neuromuscularjunction.

The term muscle cell as used herein refers to any cell which contributesto muscle tissue. Myoblasts, satellite cells, myotubes, and myofibriltissues are all included in the term “muscle cells” and may all betreated using the methods of the invention. Muscle cell effects may beinduced within skeletal, cardiac and smooth muscles.

Mitogenesis may be induced in muscle cells, including myoblasts orsatellite cells, of skeletal muscle, smooth muscle or cardiac muscle.Mitogenesis as used herein refers to any cell division which results inthe production of new muscle cells in the patient. More specifically,mitogenesis in vitro is defined as an increase in mitotic index relativeto untreated cells of 50%, more preferably 100%, and most preferably300%, when the cells are exposed to labelling agent for a timeequivalent to two doubling times. The mitotic index is the fraction ofcells in the culture which have labelled nuclei when grown in thepresence of a tracer which only incorporates during S phase (i.e., BrdU)and the doubling time is defined as the average time required for thenumber of cells in the culture to increase by a factor of two.

An effect on mitogenesis in vivo is defined as an increase in satellitecell activation as measured by the appearance of labelled satellitecells in the muscle tissue of a mammal exposed to a tracer which onlyincorporates during S phase (i.e., BrdU). The useful therapeutic isdefined in vivo as a compound which increases satellite cell activationrelative to a control mammal by at least 10%, more preferably by atleast 50%, and most preferably by more than 200% when the mammal isexposed to labelling agent for a period of greater than 15 minutes andtissues are assayed between 10 hours and 24 hours after administrationof the mitogen at the therapeutic dose. Alternatively, satellite cellactivation in vivo may be detected by monitoring the appearance of theintermediate filament vimentin by immunological or RNA analysis methods.When vimentin is assayed, the useful mitogen is defined as one whichcauses expression of detectable levels of vimentin in the muscle tissuewhen the therapeutically useful dosage is provided.

Myogenesis as used herein refers to any fusion of myoblasts to yieldmyotubes. Most preferably, an effect on myogenesis is defined as anincrease in the fusion of myoblasts and the enablement of the muscledifferentiation program. The useful myogenic therapeutic is defined as acompound which confers any increase in the fusion index in vitro. Morepreferably, the compound confers at least a 2.0-fold increase and, mostpreferably, the compound confers a 3-fold or greater increase in thefusion index relative to the control. The fusion index is defined as thefraction of nuclei present in multinucleated cells in the culturerelative to the total number of nuclei present in the culture. Thepercentages provided above are for cells assayed after 6 days ofexposure to the myogenic compound and are relative to an untreatedcontrol. Myogenesis may also be determined by assaying the number ofnuclei per area in myotubes or by measurement of the levels of musclespecific protein by Western analysis. Preferably, the compound confersat least a 2.0-fold increase in the density of myotubes using the assayprovided, for example, herein, and, most preferably, the compoundconfers a 3-fold or greater increase.

The growth of muscle may occur by the increase in the fiber size and/orby increasing the number of fibers. The growth of muscle as used hereinmay be measured by A) an increase in wet weight, B) an increase inprotein content, C) an increase in the number of muscle fibers, or D) anincrease in muscle fiber diameter. An increase in growth of a musclefiber can be defined as an increase in the diameter where the diameteris defined as the minor axis of ellipsis of the cross section. Theuseful therapeutic is one which increases the wet weight, proteincontent and/or diameter by 10% or more, more preferably by more than 50%and most preferably by more than 100% in an animal whose muscles havebeen previously degenerated by at least 10% and relative to a similarlytreated control animal (i.e., an animal with degenerated muscle tissuewhich is not treated with the muscle growth compound). A compound whichincreases growth by increasing the number of muscle fibers is useful asa therapeutic when it increases the number of fibers in the diseasedtissue by at least 1%, more preferably at least 20%, and mostpreferably, by at least 50%. These percentages are determined relativeto the basal level in a comparable untreated undiseased mammal or in thecontralateral undiseased muscle when the compound is administered andacts locally.

The survival of muscle fibers as used herein refers to the prevention ofloss of muscle fibers as evidenced by necrosis or apoptosis or theprevention of other mechanisms of muscle fiber loss. Survival as usedherein indicates an decrease in the rate of cell death of at least 10%,more preferably by at least 50%, and most preferably by at least 300%relative to an untreated control. The rate of survival may be measuredby counting cells stainable with a dye specific for dead cells (such aspropidium iodide) in culture when the cells are 8 dayspost-differentiation (i.e., 8 days after the media is changed from 20%to 0.5% serum).

Muscle regeneration as used herein refers to the process by which newmuscle fibers form from muscle progenitor cells. The useful therapeuticfor regeneration confers an increase in the number of new fibers by atleast 1%, more preferably by at least 20%, and most preferably by atleast 50%, as defined above.

The differentiation of muscle cells as used herein refers to theinduction of a muscle developmental program which specifies thecomponents of the muscle fiber such as the contractile apparatus (themyofibril). The therapeutic useful for differentiation increases thequantity of any component of the muscle fiber in the diseased tissue byat least 10% or more, more preferably by 50% or more, and mostpreferably by more than 100% relative to the equivalent tissue in asimilarly treated control animal.

Atrophy of muscle as used herein refers to a significant loss in musclefiber girth. By significant atrophy is meant a reduction of muscle fiberdiameter in diseased, injured or unused muscle tissue of at least 10%relative to undiseased, uninjured, or normally utilized tissue.

Methods for treatment of diseases or disorders using the polypeptides orother compounds described herein are also part of the invention.Examples of muscular disorders which may be treated include skeletalmuscle diseases and disorders such as myopathies, dystrophies, myoneuralconductive diseases, traumatic muscle injury, and nerve injury. Cardiacmuscle pathologies such as cardiomyopathies, ischemic damage, congenitaldisease, and traumatic injury may also be treated using the methods ofthe invention, as may smooth muscle diseases and disorders such asarterial sclerosis, vascular lesions, and congenital vascular diseases.For example, Duchenne's muscular dystrophy, Becker's dystrophy, andMyasthenia gravis are but three of the diseases which may be treatedusing the methods of the invention.

The invention also includes methods for the prophylaxis or treatment ofa tumor of muscle cell origin such as rhabdomyosarcoma. These methodsinclude administration of an effective amount of a substance whichinhibits the binding of one or more of the polypeptides described hereinand inhibiting the proliferation of the cells which contribute to thetumor.

The methods of the invention may also be used to treat a patientsuffering from a disease caused by a lack of a neurotrophic factor. Bylacking a neurotrophic factor is meant a decreased amount ofneurotrophic factor relative to an unaffected individual sufficient tocause detectable decrease in neuromuscular connections and/or muscularstrength. The neurotrophic factor may be present at levels 10% belowthose observed in unaffected individuals. More preferably, the factor ispresent at levels 20% lower than are observed in unaffected individuals,and most preferably the levels are lowered by 80% relative to unaffectedindividuals under similar circumstances.

The methods of the invention make use of the fact that the neuregulinproteins are encoded by the same gene. A variety of messenger RNAsplicing variants (and their resultant proteins) are derived from thisgene and many of these products show binding to P185^(ebB2) andactivation of the same. Products of this gene have been used to showmuscle cell mitogenic activity (see Examples 1 and 2, below),differentiation (Examples 3 and 6), and survival (Examples 4 and 5).This invention provides a use for all of the known products of theneuregulin gene (described herein and in the references listed above)which have the stated activities as muscle cell mitogens,differentiation factors, and survival factors. Most preferably,recombinant human GGF2 (rhGGF2) is used in these methods.

The invention also relates to the use of other, not yet naturallyisolated, splicing variants of the neuregulin gene. FIG. 29 shows theknown patterns of splicing. These patterns are derived from polymerasechain reaction experiments (on reverse transcribed RNA), analysis ofcDNA clones (as presented within), and analysis of published sequencesencoding neuregulins (Peles et al., Cell 69:205 (1992) and Wen et al.,Cell 69:559 (1992)). These patterns, as well as additional patternsdisclosed herein, represent probable splicing variants which exist. Thesplicing variants are fully described in Goodearl et al., U.S. Ser. No.08/036,555, filed Mar. 24, 1993, incorporated herein by reference.

More specifically, cell division, survival, differentiation and growthof muscle cells may be achieved by contacting muscle cells with apolypeptide defined by the formulaWYBAZCX (SEQ ID NOS: 212–379)

-   -   wherein WYBAZCX is composed of the polypeptide segments shown in        FIG. 30 (SEQ ID NOS: 185–211) wherein W comprises the        polypeptide segment F (SEQ ID NO: 206), or is absent wherein Y        comprises the polypeptide segment E (SEQ ID NO: 207), or is        absent; wherein Z comprises the polypeptide segment G (SEQ ID        NO: 210) or is absent; wherein X comprises the polypeptide        segment C/D HKL (SEQ ID NO: 185), C/D H (SEQ ID NO: 186), C/D HL        (SEQ ID NO: 187), C/D D (SEQ ID NO: 188), C/D′ HL (SEQ ID NO:        189), C/D′ HKL (SEQ ID NO: 190), C/D′ H (SEQ ID NO: 191), C/D′ D        (SEQ ID NO: 192), C/D C/D′ HKL (SEQ ID NO: 193), C/D C/D′ H (SEQ        ID NO: 194), C/D C/D′ HL (SEQ ID NO: 195), C/D C/D′ D (SEQ ID        NO: 196), C/D D′ H (SEQ ID NO: 197), C/D D′ HL (SEQ ID NO: 198),        C/D D′ HKL (SEQ ID NO: 199), C/D′ D′ H (SEQ ID NO: 200), C/D′ D′        HL (SEQ ID NO: 201), C/D′ D′ HKL (SEQ ID NO: 202), C/D C/D′ D′ H        (SEQ ID NO: 203), C/D C/D′ D′ HL (SEQ ID NO: 204), or C/D C/D′        D′ HKL (SEQ ID NO: 205).

Furthermore, the invention includes a method of treating muscle cells bythe application to the muscle cell of a

-   -   −30 kD polypeptide factor isolated from the MDA-MB 231 human        breast cell line; or    -   −35 kD polypeptide factor isolated from the rat I-EJ transformed        fibroblast cell line to the glial cell or    -   −75 kD polypeptide factor isolated from the SKBR-3 human breast        cell line; or    -   −44 kD polypeptide factor isolated from the rat I-EJ transformed        fibroblast cell line; or    -   −25 kD polypeptide factor isolated from activated mouse        peritoneal macrophages; or    -   −45 kD polypeptide factor isolated from the MDA-MB 231 human        breast cell; or    -   −7 to 14 kD polypeptide factor isolated from the ATL-2 human        T-cell line to the glial cell; or    -   −25 kD polypeptide factor isolated from the bovine kidney cells;        or    -   −42 kD ARIA polypeptide factor isolated from brain;    -   −46–47 kD polypeptide factor which stimulates 0–2A glial        progenitor cells; or    -   −43–45 kD polypeptide factor, GGFIII,175

U.S. patent application Ser. No. 07/931,041, filed Aug. 17, 1992,incorporated herein by reference.

The invention further includes methods for the use of the EGFL1, EGFL2,EGFL3, EGFL4, EGFL5, and EGFL6 polypeptides, FIGS. 37 to 42 and SEQ IDNos. 150 to 155, respectively, for the treatment of muscle cells in vivoand in vitro.

Also included in the invention is the administration of the GGF2polypeptide whose sequence is shown in FIG. 44 for the treatment ofmuscle cells.

An additional important aspect of the invention are methods for treatingmuscle cells using:

-   -   (a) a basic polypeptide factor also known to have glial cell        mitogenic activity, in the presence of fetal calf plasma, a        molecular weight of from about 30 kD to about 36 kD, and        including within its amino acid sequence any one or more of the        following peptide sequences:    -   F K G D A H T E (SEQ ID NO: 1)    -   A S L A D E Y E Y M X K (SEQ ID NO: 2)    -   T E T S S S G L X L K (SEQ ID NO: 3)    -   A S L A D E Y E Y M R K (SEQ ID NO: 7)    -   A G Y F A E X A R (SEQ ID NO: 11)    -   T T E M A S E Q G A (SEQ ID NO:13)    -   A K E A L A A L K (SEQ ID NO: 14)    -   F V L Q A K K (SEQ ID NO: 15)    -   E T Q P D P G Q I L K K V P M V I G A Y T (SEQ ID NO: 165)    -   E Y K C L K F K W F K K A T V M (SEQ ID NO: 17)    -   E X K F Y V P (SEQ ID NO: 19)    -   K L E F L X A K (SEQ ID NO: 32); and    -   (b) a basic polypeptide factor for use in treating muscle cells        which is also known to stimulate glial cell mitogenesis in the        presence of fetal calf plasma, has a molecular weight of from        about 55 kD to about 63 kD, and including within its amino acid        sequence any one or more of the following peptide sequences:    -   V H Q V W A A K (SEQ ID NO: 45)    -   Y I F F M E P E A X S S G (SEQ ID NO: 46)    -   L G A W G P P A F P V X Y (SEQ ID NO: 47)    -   W F V V I E G K (SEQ ID NO: 48)    -   A S P V S V G S V Q E L Q R (SEQ ID NO: 49)    -   V C L L T V A A L P P T (SEQ ID NO: 50)    -   K V H Q V W A A K (SEQ ID NO: 51)    -   K A S L A D S G E Y M X K (SEQ ID NO: 49)    -   D L L L X V (SEQ ID NO: 53)

Methods for the use of the peptide sequences set out above, derived fromthe smaller molecular weight polypeptide factor, and from the largermolecular weight polypeptide factor, are also aspects of this invention.Monoclonal antibodies to the above peptides are themselves usefulinvestigative tools and therapeutics.

Thus, the invention further embraces methods of using a polypeptidefactor having activities useful for treating muscle cells and includingan amino acid sequence encoded by:

-   -   (a) a DNA sequence shown in any one of FIGS. 27A, 27B or 27C,        SEQ ID Nos. 129–131, respectively;    -   (b) a DNA sequence shown in FIG. 21, SEQ ID No. 85;    -   (c) the DNA sequence represented by nucleotides 281–557 of the        sequence shown in FIG. 27A, SEQ ID No. 129; or    -   (d) a DNA sequence hybridizable to any one of the DNA sequences        according to (a), (b) or (c).

Following factors as muscle cell mitogens:

-   -   (a) a basic polypeptide factor which has, if obtained from        bovine pituitary material, an observed molecular weight, whether        in reducing conditions or not, of from about 30 kD to about 36        kD on SDS-polyacrylamide gel electrophoresis which factor has        muscle cell mitogenic activity including stimulating the        division of myoblasts, and when isolated using reversed-phase        HPLC retains at least 50% of said activity after 10 weeks        incubation in 0.1% trifluoroacetic acid at 4° C.; and    -   (b) a basic polypeptide factor which has, if obtained from        bovine pituitary material, an observed molecular weight, under        non-reducing conditions, of from about 55 kD to about 63 Kd on        SDS-polyacrylamide gel electrophoresis which factor the human        equivalent of which is encoded by DNA clone GGF2HBS5 and which        factor has muscle cell mitogenic activity and when isolated        using reversed-phase HPLC retains at least 50% of the activity        after 4 days incubation in 0.1% trifluoroacetic acid at 4° C.

Thus other important aspects of the invention are the use of:

-   -   (a) A series of human and bovine polypeptide factors having cell        mitogenic activity including stimulating the division of muscle        cells. These peptide sequences are shown in FIGS. 30, 31, 32 and        33, SEQ ID Nos. 132–133, respectively.    -   (b) A series of polypeptide factors having cell mitogenic        activity including stimulating the division of muscle cells and        purified and characterized according to the procedures outlined        by Lupu et al. Science 249: 1552 (1990); Lupu et al. Proc. Natl.        Acad. Sci USA 89: 2287 (1992); Holmes et al. Science 256: 1205        (1992); Peles et al. 69: 205 (1992); Yarden and Peles        Biochemistry 30: 3543 (1991); Dobashi et al. Proc. Natl. Acad.        Sci. 88: 8582 (1991); Davis et al. Biochem. Biophys. Res.        Commun. 179: 1536 (1991); Beaumont et al., patent application        PCT/US91/03443 (1990); Bottenstein, U.S. Pat. No. 5,276,145,        issued Jan. 4, 1994; and Greene et al. patent application        PCT/US91/02331 (1990).    -   (c) A polypeptide factor (GGFBPP5) having glial cell mitogenic        activity including stimulating the division of muscle cells. The        amino acid sequence is shown in FIG. 31, SEQ ID No. 144.

Methods for stimulating mitogenesis of a myoblast by contacting themyoblast cell with a polypeptide defined above as a muscle cell mitogenin vivo or in vitro are included as features of the invention.

Muscle cell treatments may also be achieved by administering DNAencoding the polypeptide compounds described above in an expressiblegenetic construction. DNA encoding the polypeptide may be administeredto the patient using techniques known in the art for delivering DNA tothe cells. For example, retroviral vectors, electroporation or liposomesmay be used to deliver DNA.

The invention includes the use of the above named family of proteins asextracted from natural sources (tissues or cell lines) or as prepared byrecombinant means.

Other compounds in particular, peptides, which bind specifically to thep185^(erbB2) receptor can also be used according to the invention asmuscle cell mitogens. A candidate compound can be routinely screened forp185^(erbB2) binding, and, if it binds, can then be screened for glialcell mitogenic activity using the methods described herein.

The invention includes use of any modifications or equivalents of theabove polypeptide factors which do not exhibit a significantly reducedactivity. For example, modifications in which amino acid content orsequence is altered without substantially adversely affecting activityare included. The statements of effect and use contained herein aretherefore to be construed accordingly, with such uses and effectsemploying modified or equivalent factors being part of the invention.

The human peptide sequences described above and presented in FIGS. 30,31, 32 and 33 (SEQ ID Nos. 386, 388, 389, 391–413) respectively,represent a series of splicing variants which can be isolated as fulllength complementary DNAs (cDNAS) from natural sources (cDNA librariesprepared from the appropriate tissues) or can be assembled as DNAconstructs with individual exons (e.g., derived as separate exons) bysomeone skilled in the art.

The invention also includes a method of making a medicament for treatingmuscle cells, i.e., for inducing muscular mitogenesis, myogenesis,differentiation, or survival, by administering an effective amount of apolypeptide as defined above. Such a medicament is made by administeringthe polypeptide with a pharmaceutically effective carrier.

Another aspect of the invention is the use of a pharmaceutical orveterinary formulation comprising any factor as defined above formulatedfor pharmaceutical or veterinary use, respectively, optionally togetherwith an acceptable diluent, carrier or excipient and/or in unit dosageform. In using the factors of the invention, conventional pharmaceuticalor veterinary practice may be employed to provide suitable formulationsor compositions.

Thus, the formulations to be used as a part of the invention can beapplied to parenteral administration, for example, intravenous,subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular,intracranial, intracapsular, intraspinal, intracisternal,intraperitoneal, topical, intranasal, aerosol, scarification, and alsooral, buccal, rectal or vaginal administration.

The formulations of this invention may also be administered by thetransplantation into the patient of host cells expressing the DNAencoding polypeptides which are effective for the methods of theinvention or by the use of surgical implants which release theformulations of the invention.

Parenteral formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are to be foundin, for example, “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain as excipientssterile water or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, or hydrogenated naphthalenes,biocompatible, biodegradable lactide polymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the present factors. Other potentially useful parenteraldelivery systems for the factors include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain as excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel to be applied intranasally. Formulations for parenteraladministration may also include glycocholate for buccal administration,methoxysalicylate for rectal administration, or citric acid for vaginaladministration.

The present factors can be used as the sole active agents, or can beused in combination with other active ingredients, e.g., other growthfactors which could facilitate neuronal survival in neurologicaldiseases, or peptidase or protease inhibitors.

The concentration of the present factors in the formulations of theinvention will vary depending upon a number of issues, including thedosage to be administered, and the route of administration.

In general terms, the factors of this invention may be provided in anaqueous physiological buffer solution containing about 0.1 to 10% w/vcompound for parenteral administration. General dose ranges are fromabout 1 mg/kg to about 1 g/kg of body weight per day; a preferred doserange is from about 0.01 mg/kg to 100 mg/kg of body weight per day. Thepreferred dosage to be administered is likely to depend upon the typeand extent of progression of the pathophysiological condition beingaddressed, the overall health of the patient, the make up of theformulation, and the route of administration.

The polypeptide factors utilized in the methods of the invention canalso be used as immunogens for making antibodies, such as monoclonalantibodies, following standard techniques. These antibodies can, inturn, be used for therapeutic or diagnostic purposes. Thus, conditionsperhaps associated with muscle diseases resulting from abnormal levelsof the factor may be tracked by using such antibodies. In vitrotechniques can be used, employing assays on isolated samples usingstandard methods. Imaging methods in which the antibodies are, forexample, tagged with radioactive isotopes which can be imaged outsidethe body using techniques for the art of tumor imaging may also beemployed.

A further general aspect of the invention is the use of a factor of theinvention in the manufacture of a medicament, preferably for thetreatment of a muscular disease or disorder. The “GGF2” designation isused for all clones which were previously isolated with peptide sequencedata derived from GGF-II protein (i.e., GGF2HBS5, GGF2BPP3) and, whenpresent alone (i.e., GGF2 or rhGGF2), to indicate recombinant humanprotein encoded by plasmids isolated with peptide sequence data derivedfrom the GGF-II protein (i.e., as produced in insect cells from theplasmid HBS5). Recombinant human GGF from the GGFHBS5 clone is calledGGF2, rhGGF2 and GGF2HBS5 polypeptide.

Treating as used herein means any administration of the compoundsdescribed herein for the purpose of increasing muscle cell mitogenesis,survival, and/or differentiation, and/or decreasing muscle atrophy anddegeneration. Most preferably, the treating is for the purpose ofreducing or diminishing the symptoms or progression of a disease ordisorder of the muscle cells. Treating as used herein also means theadministration of the compounds for increasing or altering the musclecells in healthy individuals. The treating may be brought about by thecontacing of the muscle cells which are sensitive or responsive to thecompounds described herein with an effective amount of the compound, asdescribed above. Inhibitors of the compounds described herein may alsobe used to halt or slow diseases of muscle cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings will first be described.

Drawings

FIG. 1 is a graph showing the results of rhGGF2 in a myoblastmitogenesis assay.

FIG. 2 is a graph showing the effect of rhGGF2 on the number of nucleiin myotubes.

FIG. 3 is a graph of a survival assay showing the effect of rhGGF2 onsurvival of differentiated myotubes.

FIG. 4 is a graph of survival assays showing the effect of rhGGF2 ondifferentiated myotubes relative to human platelet derived growthfactor, human fibroblast growth factor, human epidermal growth factor,human leucocyte inhibitory factor, and human insulin-like growth factorsI and II.

FIG. 5 is a graph showing the increased survival on Duchenne musculardystrophy cells in the presence of rhGGF2.

FIG. 6 is a graph of increasing human growth hormone (hGH) expression inC2 cells from an hGH reporter gene under control of the AchR deltasubunit transcriptional control elements. This increase is tied to theaddition of GGF2 to the media.

FIG. 7 is a graph of increasing hGH reporter synthesis and bungarotoxin(BTX) binding to AchRs following the addition of increasing amounts ofGGF2 to C2 cells.

FIGS. 8, 9, 10 and 11 are the peptide sequences derived from GGF-I andGGF-II, SEQ ID Nos. 1–20, 22–29, 32–50 and 165, (see Examples 11–13hereinafter).

FIG. 8 shows the 21 peptide sequences (SEQ ID Nos 1–20, and 169)obtained from lysyl endopeptidase and protease V8 digestion of purifiedbovine pituitary GGF-I.

FIG. 9, Panel A, is the sequences of GGF-I peptides used to designdegenerate oligonucleotide probes and degenerate PCR primers are listed(SEQ ID Nos. 1, 17 and 22–29). Some of the sequences in Panel A werealso used to design synthetic peptides. Panel B is a listing of thesequences of novel peptides that were too short (less than 6 aminoacids) for the design of degenerate probes or degenerate PCR primers(SEQ ID Nos. 17 and 32);

FIG. 10 shows various trypsin and lysyl endopeptidase C are peptidesequences derived from GGF-II, SEQ ID Nos. 33–39, 164–166, 51–52.

FIG. 11, Panel A, is a listing of the sequences of GGF-II peptides usedto design degenerate oligonucleotide probes and degenerate PCR primers(SEQ ID Nos. 45–52). Some of the sequences in Panel A were used todesign synthetic peptides. Panel B is a listing of the novel peptidethat was too short (less than 6 amino acids) for the design ofdegenerate probes or degenerate PCR primers (SEQ ID No. 53);

FIGS. 12, 13A, 13B, 14, 15, 16, 17, 18, and 19 relate to Example 8,below, and depict the mitogenic activity of factors of the invention;

FIG. 12 shows a graph comparing BrUdR-ELISA and [¹²⁵I]UdR countingmethods for the DNA synthesis assay in Schwann cell cultures.

FIGS. 13A and 13B show graphs comparing Br-UdR immunoreactivity with thenumber of Br-UdR labeled cells.

FIG. 14 shows the mitogenic response of rat sciatic nerve Schwann cellsto GGFs.

FIG. 15 shows a graph quantifying DNA synthesis in rat sciatic nerveSchwann cells and 3T3 fibroblasts in the presence of GGFs.

FIG. 16 shows a graph of the mitogenic response of BHK 21 C13 cells toFCS and GGFs.

FIG. 17 shows a graph of survival and proliferation of BH 21 C13 cellmicro cultures after 48 hours in the presence of GGFs.

FIG. 18 shows a graph of the mitogenic response of C6 cells to FCS.

FIGS. 19A and 19B are graphs showing the mitogenic response of C6 cellsto aFGF and GGFs.

FIGS. 20, 21, 22, 23, 24, 25, 26, and 27 relate to Example 10, below andare briefly described below:

FIG. 20 is a listing of the degenerate oligonucleotide probes (SEQ IDNos. 54–76, 78–88) designed from the novel peptide sequences in FIG. 7,Panel A and FIG. 9, Panel A;

FIG. 21 depicts a stretch of the putative bovine GGF-II gene sequencefrom the recombinant 30 bovine genomic phage GGF2BG1, containing thebinding site of degenerate oligonucleotide probes 609 and 650 (see FIG.20, SEQ ID NOs. 66 and 69, respectively). The figure is the codingstrand of the DNA sequence (SEQ ID NO. 89) and the deduced amino acidsequence (SEQ ID NO: 385) in the third reading frame. The sequence ofpeptide 12 from factor 2 (bold) is part of a 66 amino acid open readingframe (nucleotides 75272);

FIG. 22A shows is the degenerate PCR primers SEQ ID Nos. 86–104) andFIG. 22B shows the unique PCR primers (SEQ ID Nos. 105–115) used inexperiments to isolate segments of the bovine GGF-II coding sequencespresent in RNA from posterior pituitary;

FIG. 23 depicts of the nine distinct contiguous bovine GGF-II cDNAstructures and sequences that were obtained in PCR amplificationexperiments. The top line of the Figure is a schematic of the codingsequences which contribute to the cDNA structures that werecharacterized;

FIG. 24 is a physical map of bovine recombinant phage of GGF2BG1. Thebovine fragment is roughly 20 kb in length and contains two exons (bold)of the bovine GGF-II gene. Restriction sites for the enzymes Xbal, SpeI,Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map.Shaded portions correspond to fragments which were subcloned forsequencing;

FIG. 25 is a schematic of the structure of three alternative geneproducts of the putative bovine GGF-II gene. Exons are listed A throughE in the order of their discovery. The alternative splicing patterns 1,2 and 3 generate three overlapping deduced protein structures (GGF2BPP1,2, and 3), which are displayed in the various FIGS. 27A, 27B, 27C(described below);

FIG. 26 (SEQ ID Nos. 116–128 45, 52, and 53) is a comparison of theGGF-I and GGF-II sequences identified in the deduced protein sequencesshown in FIGS. 27A, 27B, 27C (described below) with the novel peptidesequences listed in FIGS. 9 and 11. The Figure shows that six of thenine novel GGF-II peptide sequences are accounted for in these deducedprotein sequences. Two peptide sequences similar to GGF-I sequences arealso found;

FIG. 27 FIG. 27A is a listing of the coding strand DNA sequence (SEQ IDNO: 133) and deduced amino acid sequence (SEQ ID NO:384) of the cDNAobtained from splicing pattern number 1 in FIG. 25. This partial cDNA ofthe putative bovine GGF-II gene encodes a protein of 206 amino acids inlength. Peptides in bold were those identified from the lists presentedin FIGS. 9 and 11. Potential glycosylation sites are underlined (alongwith polyadenylation signal AATAAA) (SEQ ID NO:420);

FIGS. 27(B–C) is a listing of the coding strand DNA sequence (SEQ ID NO:134) and deduced amino acid sequence SEQ ID NO:385 of the cDNA obtainedfrom splicing pattern number 2 in FIG. 25. This partial cDNA of theputative bovine GGF-II gene encodes a protein of 281 amino acids inlength. Peptides in bold are those identified from the lists presentedin FIGS. 7 and 9. Potential glycosylation sites are underlined (alongwith polyadenylation signal AATAAA) SEQ ID NO: 420;

FIGS. 27(D–E) is a listing of the coding strand DNA sequence and deducedamino acid sequence SEQ ID NO: 387 of the cDNA obtained from splicingpattern number 3 in FIG. 25. This partial cDNA of the putative bovineGGF-II gene encodes a protein of 257 amino acids in length. Peptides inbold are those identified from the lists in FIGS. 9 and 11. Potentialglycosylation sites are underlined (along with polyadenylation signalAATAAA) SEQ ID NO: 420.

FIG. 28, which relates to Example 15 hereinafter, is an autoradiogram ofa cross hybridization analysis of putative bovine GGF-II gene sequencesto a variety of mammalian DNAs on a southern blot. The filter containslanes of EcoRI-digested DNA (5 μg per lane) from the species listed inthe Figure. The probe detects a single strong band in each DNA sample,including a four kilobase fragment in the bovine DNA as anticipated bythe physical map in FIG. 24. Bands of relatively minor intensity areobserved as well, which could represent related DNA sequences. Thestrong hybridizing band from each of the other mammalian DNA samplespresumably represents the GGF-II homologue of those species.

FIG. 29 is a diagram of representative splicing variants. The codingsegments are represented by F, E, B, A, G, C, C/D, C/D′, D, D′, H, K andL. The location of the peptide sequences derived from purified proteinare indicated by “o”.

FIGS. 30(A–R) is a listing of the DNA sequences and predicted peptidesequences (SEQ ID NOS. 391–413 of the coding segments of GGF. Line 1 isa listing of the predicted amino acid sequences of bovine GGF, line 2 isa listing of the nucleotide sequences of bovine GGF, line 3 is a listingof the nucleotide sequences of human GGF (heregulin) (nucleotide basematches are indicated with a vertical line) and line 4 is a listing ofthe predicted amino acid sequences of human GGF/heregulin where itdiffers from the predicted bovine sequence. Coding segments E, A′ and Krepresent only the bovine sequences. Coding segment D′ represents onlythe human (heregulin) sequence.

FIGS. 31(A–B) is the predicted GGF2 amino acid sequence and nucleotidesequence of BPP5(SEQ ID No: 389 and SEQ ID No: 148 respectively). Theupper line is the nucleotide sequence and the lower line is thepredicted amino acid sequence.

FIGS. 32(A–B) is the predicted amino acid sequence and nucleotidesequence of GGF2BPP2(SEQ ID No:149 and SEQ ID No: 386, respectively).The upper line is the nucleotide sequence and the lower line is thepredicted amino acid sequence.

FIGS. 33(A–C) (SEQ ID No. 146) is the predicted amino acid sequence andnucleotide sequence of GGF2BPP4(SEQ ID No: 388 and SEQ ID No: 150respectively). The upper line is the nucleotide sequence and the lowerline is the predicted amino acid sequence.

FIG. 34 (SEQ ID Nos. 147–149) depicts the alignment of two GGF peptidesequences (GGF2BPP4 and GGF2BPP5) with the human EGF (hEGF). Asterisksindicate positions of conserved cysteines.

FIG. 35 depicts the level of GGF activity (Schwann cell mitogenic assay)and tyrosine phosphorylation of a ca. 200 kD protein (intensity of a 200kD band on an autoradiogram of a Western blot developed with anantiphosphotyrosine polyclonal antibody) in response to increasingamounts of GGF.

FIGS. 36(A–B) is a list of splicing variants derived from the sequencesshown in FIGS. 30(A–R).

FIG. 37 is the predicted amino acid sequence, bottom SEQ ID NO:414 andnucleic sequence, top, of EGFL1 (SEQ ID No. 150).

FIG. 38 is the predicted amino acid sequence, bottom (SEQ ID NO:415) andnucleic sequence, top, of EGFL2 (SEQ ID No. 151).

FIG. 39 is the predicted amino acid sequence, bottom (SEQ ID NO:416) andnucleic sequence, top, of EGFL3 (SEQ ID No. 152).

FIG. 40 is the predicted amino acid sequence, bottom (SEQ ID NO: 417)and nucleic sequence, top, of EGFL4 (SEQ ID No. 153).

FIG. 41 is the predicted amino acid sequence, bottom (SEQ ID NO:418) andnucleic sequence, top, of EGFL5 (SEQ ID No. 154).

FIG. 42 is the predicted amino acid sequence, bottom (SEQ ID NO:419) andnucleic sequence, top, of EGFL6 (SEQ ID No: 155).

FIG. 43 is a scale coding segment map of the clone. T3 refers to thebacteriophage promoter used to produce mRNA from the clone. R=flankingEcoRI restriction enzyme sites. 5′ UT refers to the 5′ untranslatedregion. E, B, A, C, C/D′, and D refer to the coding segments. O=thetranslation start site. Λ=the 5′ limit of the region homologous to thebovine E segment (see Example 16) and 3′ UT refers to the 3′untranslated region.

FIGS. 44(A–D) is the predicted amino acid sequence (middle) (SEQ IDNO:17) and nucleic sequence (top) of GGF2HBS5 (SEQ ID No. 21). Thebottom (intermittent) sequence represents peptide sequences derived fromGGF-II preparations (see FIGS. 8, 9).

FIG. 45 (A) is a graph showing the purification of rGGF on cationexchange column by fraction; FIGS. 45(B–C) (B) is a photograph of aWestern blot using fractions as depicted in (A) and a GGFII specificantibody.

FIG. 46 is the sequence of the GGFHBS5, GGFHFB1 and GGFBPP5 polypeptides(SEQ ID NOS: 166, 167, and 168 respectively).

FIG. 47 is a map of the plasmid pcDHRFpolyA.

DETAILED DESCRIPTION

The invention pertains to the use of isolated and purified neuregulinfactors and DNA sequences encoding these factors, regulatory compoundswhich increase the extramuscular concentrations of these factors, andcompounds which are mimetics of these factors for the induction ofmuscle cell mitogenesis, differentiation, and survival of the musclecells in vivo and in vitro.

It is evident that the gene encoding GGF/p185^(erbB2) binding neuregulinproteins produces a number of variably-sized, differentially-spliced RNAtranscripts that give rise to a series of proteins. These proteins areof different lengths and contain some common peptide sequences and someunique peptide sequences. The conclusion that these factors are encodedby a single gene is supported by the differentially-spliced RNAsequences which are recoverable from bovine posterior pituitary andhuman breast cancer cells (MDA-MB-231)). Further support for thisconclusion derives from the size range of proteins which act as bothmitogens for muscle tissue (as disclosed herein) and as ligands for thep185^(erbB2) receptor (see below).

Further evidence to support the fact that the genes encodingGGF/p185^(erbB2) binding proteins are homologous comes from nucleotidesequence comparison. Holmes et al., (Science 256:1205–1210, 1992)demonstrate the purification of a 45-kilodalton human protein(Heregulin-α) which specifically interacts with the receptor proteinp185^(erbB2). Peles et al. (Cell 69:205 (1992)) and Wen et al. (Cell69:559 (1992)) describe a complementary DNA isolated from rat cellsencoding a protein called “neu differentiation factor” (NDF). Thetranslation product of the NDF cDNA has p185^(erbB2) binding activity.Several other groups have reported the purification of proteins ofvarious molecular weights with p185^(erbB2) binding activity. Thesegroups include Lupu et al. ((1992) Proc. Natl. Acad. Sci. USA 89:2287);Yarden and Peles ((1991) Biochemistry 30:3543); Lupu et al. ((1990)Science 249:1552)); Dobashi et al. ((1991) Biochem. Biophys. Res. Comm.179:1536); and Huang et al. ((1992) J. Biol. Chem. 257:11508–11512).

We have found that p185^(erbB2) receptor binding proteins stimulatemuscle cell mitogenesis and hence, stimulates myotube formation(myogenesis). This stimulation results in increased formation ofmyoblasts and increased formation of myotubes (myogenesis). Thecompounds described herein also stimulate increased muscle growth,differentiation, and survival of muscle cells. These ligands include,but are not limited to the GGF's, the neuregulins, the heregulins, NDF,and ARIA. As a result of this mitogenic activity, these proteins, DNAencoding these proteins, and related compounds may be administered topatients suffering from traumatic damage or diseases of the muscletissue. It is understood that all methods provided for the purpose ofmitogenesis are useful for the purpose of myogenesis. Inhibitors ofthese ligands (such as antibodies or peptide fragments) may beadministered for the treatment of muscle derived tumors.

These compounds may be obtained using the protocols described herein(Examples 9–17) and in Holmes et al., Science 256: 1205 (1992); Peles etal., Cell 69:205 (1992); Wen et al., Cell 69:559 (1992); Lupu et al.,Proc. Natl. Acad. Sci. USA 89:2287 (1992); Yarden and Peles,Biochemistry 30:3543 (1991); Lupu et al., Science 249:1552 (1990);Dobashi et al., Biochem. Biophys. Res. Comm. 179:1536 (1991); Huang etal., J. Biol. Chem. 257:11508–11512 (1992); Marchionni et al. Nature362:313, (1993); and in the GGF-III application (U.S. Ser. No.07/931,041), all of which are incorporated herein by reference. Thesequences are provided and the characteristics described for many ofthese compounds. For sequences see FIGS. 8–11, 20–27C, 29–34, 36–44, and46. For protein characteristics see FIGS. 12–19, 28 35, 45A and 45B.

Compounds may be assayed for their usefulness in vitro using the methodsprovided in the examples below. In vivo testing may be performed asdescribed in Example 1 and in Sklar et al., In Vitro Cellular andDevelopmental Biology 27A:433–434, 1991.

OTHER EMBODIMENTS

The invention includes methods for the use of any protein which issubstantial homologous to the coding segments in FIG. 30 (SEQ ID NOS:132–143, 156, and 157) as well as other naturally occurring GGFpolypeptides for the purpose of inducing muscle mitogenesis. Alsoincluded are the use of: allelic variations; natural mutants; inducedmutants; proteins encoded by DNA that hybridizes under high or lowstringency conditions to a nucleic acid naturally occurring (fordefinitions of high and low stringency see Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1989, 6.3.1–6.3.6,hereby incorporated by reference); and the use of polypeptides orproteins specifically bound by antisera to GGF polypeptides. The termalso includes the use of chimeric polypeptides that include the GGFpolypeptides comprising sequences from FIG. 28 for the induction ofmuscle mitogenesis.

As will be seen from Example 8, below, the present factors exhibitmitogenic activity on a range of cell types. The general statements ofinvention above in relation to formulations and/or medicaments and theirmanufacture should clearly be construed to include appropriate productsand uses.

A series of experiments follow which provide additional basis for theclaims described herein. The following examples relating to the presentinvention should not be construed as specifically limiting theinvention, or such variations of the invention, now known or laterdeveloped.

The examples illustrate our discovery that recombinant human GGF2(rhGGF2) confers several effects on primary human muscle culture. rhGGF2has significant effects in three independent biological activity assayson muscle cultures. The polypeptide increased mitogenesis as measured byproliferation of subconfluent quiescent myoblasts, increaseddifferentiation by confluent myoblasts in the presence of growth factor,and increased survival of differentiated myotubes as measured by loss ofdye exclusion and increased acetylcholine receptor synthesis. Theseactivities indicate efficacy of GGF2 and other neuregulins in inducingmuscle repair, regeneration, and prophylactic effects on muscledegeneration.

EXAMPLE 1 Mitogenic Activity of rhGGF on Myoblasts

Clone GGF2HBS5 was expressed in recombinant Baculovirus infected insectcells as described in Example 13, infra, and the resultant recombinanthuman GGF2 was added to myoblasts in culture (conditioned medium addedat 40 μl/ml). Myoblasts (057A cells) were grown to preconfluence in a 24well dish. Medium was removed and replaced with DMEM containing 0.5%fetal calf serum with or without GGF2 conditioned medium at aconcentration of 40 μl/ml. Medium was changed after 2 days and cellswere fixed and stained after 5 days. Total nuclei were counted as werethe number of nuclei in myoblasts (Table 1).

TABLE 1 Total Number Nuclei in Fusion Treatment of Nuclei/mm² MyotubesIndex Control 395 ± 28.3 204 ± 9.19 0.515 ± 0.01 GGF 40 μl/ml 636 ± 8.5 381 ± 82.7 0.591 ± 0.15GGF treated myoblasts showed an increased number of total nuclei (636nuclei) over untreated controls (395 nuclei) indicating mitogenicactivity. rhGGF2 treated myotubes had a greater number of nuclei (381nuclei) than untreated controls (204 nuclei). Thus, rhGGF2 enhances thetotal number of nuclei through proliferation and increased cellsurvival. rhGGF2 is also likely to enhance the formation of myotubes.

The mitogenic activity of rhGGF2 may be measured in vivo by giving acontinuous supply of GGF2 and [³H]thymidine to rat muscle via an osmoticmini pump. The muscle bulk is determined by wet weight after one and twoweeks of treatment. DNA replication is measured by counting labelednuclei in sections after coating for autoradiography (Sklar et al., InVitro Cellular and Developmental Biology 27A:433–434, 1991) in sham andrhGGF2-treated muscle. Denervated muscle is also examined in this ratanimal model via these methods and this method allows the assessment ofthe role of rhGGF2 in muscle atrophy and repair. Mean fiber diameter canalso be used for assessing effects of FGF on prevention of atrophy.

EXAMPLE 2

Effect of rhGGF2 on Muscle Cell Mitogenesis

Quiescent primary clonal human myoblasts were prepared as previouslydescribed (Sklar, R., Hudson, A., Brown, R., In vitro Cellular andDevelopmental Biology 1991; 27A:433–434). The quiescent cells weretreated with the indicated agents (rhGGF2 conditioned media, PDGF withand without methylprednisolone, and control media) in the presence of 10μM BrdU, 0.5% FCS in DMEM. After two days the cells were fixed in 4%paraformaldehyde in PBS for 30 minutes, and washed with 70% ethanol. Thecells were then incubated with an anti-BrdU antibody, washed, andantibody binding was visualized with a peroxidase reaction. The numberof staining nuclei were then quantified per area. The results show thatGGF2 induces an increase in the number of labelled nuclei per area overcontrols (see Table 2).

TABLE 2 Mitogenic Effects of GGF on Human Myoblasts Labelled T-TestTreatment Nuclei/cm² p value Control 120 ± 22.4 Infected Control 103 ±11.9 GGF 5 μl/ml 223 ± 33.8 0.019 PDGF 20 ng/ml 418 ± 45.8 0.0005 IGFI30 ng/ml  280 ± 109.6 0.068 Methylprednisolone 1.0 μM 142 ± 20.7 0.293Platelet derived growth factor (PDGF) was used as a positive control.Methylprednisolone (a corticosteroid) was also used in addition torhGGF2 and showed no significant increase in labelling of DNA.

rhGGF2 purified to homogeneity (>95% pure) is also mitogenic for humanmyoblasts (FIG. 1).

Recombinant human GGF2 also causes mitogenesis of primary humanmyoblasts (see Table 2 and FIG. 1). The mitogenesis assay is performedas described above. The mitotic index is then calculated by dividing thenumber of BrdU positive cells by the total number of cells.

EXAMPLE 3

Effect of rhGGF2 on Muscle Cell Differentiation

The effects of purified rhGGF2 (95% pure) on muscle culturedifferentiation were examined (FIG. 2). Confluent myoblast cultures wereinduced to differentiate by lowering the serum content of the culturemedium from 20% to 0.5%. The test cultures were treated with theindicated concentration of rhGGF2 for six days, refreshing the culturemedium every 2 days. The cultures were then fixed, stained, and thenumber of nuclei counted per millimeter. The data in FIG. 2 demonstratea large increase in the number of nuclei in myotubes when rhGGF2 ispresent, relative to controls.

EXAMPLE 4

Effect of rhGGF2 on the Survival of Differentiated Myotubes

The survival of differentiated myotubes was significantly increased byrhGGF2 treatment. Muscle cultures were differentiated in the presence ofrhGGF2 and at various times the number of dead myotubes were counted bypropidium iodide staining. As can be seen in FIG. 3, the number of deadmyotubes is lower in the rhGGF2 treated culture at 4, 5, 6, and 8 daysof differentiation. The number of nuclei in myotubes was significantlyincreased by GGF2 treatment compared to untreated cultures after 8 daysof differentiation. Specifically, the control showed 8.6 myonuclei/mm²,while rhGGF2 treated cultures showed 57.2 myonuclei/mm² (p=0.035) whencounted on the same plates after geimsa staining.

The survival assay was also performed with other growth factors whichhave known effects on muscle culture. The rhGGF2 effect was unique amongthe growth factors tested (FIG. 4). In this experiment cultures weretreated in parallel with the rhGGF2 treated plates with the indicatedconcentrations of the various growth factors. Survival of myotubes wasmeasured as above at 8 days of differentiation of 057A myoblast cells.Concentrations of factors were as follows: rhGGF2: 100 ng/ml; humanplatelet derived growth factor: 20 ng/ml; human basic fibroblast growthfactor: 25 ng/ml; human epidermal growth factor: 30 ng/ml; humanleucocyte inhibitory factor: 10 ng/ml; human insulin like growth factorI: 30 ng/ml; human insulin like growth factor II: 25 ng/ml.

The observed protection of differentiated myotubes from death indicatesthat rhGGF2 has promise as a therapy for intervention of muscledegeneration characterized by numerous muscle diseases. Thus, agentswhich increase the extramuscular concentration of neuregulins may have aprophylactic effect or slow the progress of muscle-wasting disorders andincrease rates of muscle differentiation, repair, conditioning, andregeneration.

EXAMPLE 5

rhGGF2 Promotes Survival of Differentiated Myotubes with a GeneticDefect at the Duchenne Muscular Dystrophy Locus

The positive effects of rhGGF2 on myotube survival could reflectpotential efficacy in degenerative disorders. These effects on myotubesurvival were tested on a clonally-derived primary Duchenne myoblast todetermine if the response observed in normal muscle culture could alsobe demonstrated in cultures derived from diseased individuals. The datapresented in FIG. 5 was obtained using the same muscle cultureconditions (Example 4, above) used for normal individual. rhGGF2significantly decreased the number of dead myotubes in thedifferentiated Duchenne muscle culture, compared to controls (p=0.032).Concentrations were as follows: GGF2: 100 ng/ml; human platelet derivedgrowth factor: 20 ng/ml; human insulin like growth factor I: 30 ng/ml.

This example demonstrates that rhGGF2 can also promote survival ofdifferentiated Duchenne myotubes and provides strong evidence thatrhGGF2 may slow or prevent the course of muscle degeneration and wastingin mammals.

EXAMPLE 6

rhGGF2 Effect on the Differentiation Program: Induction of MHC Slow andDystrophin Proteins

The effects of purified rhGGF2 on muscle culture differentiation wasalso examined by Western analysis of culture lysates. The levels ofmuscle specific proteins were determined in triplicate treated anduntreated cultures. These cultures were prepared and treated as aboveexcept that the plate size was increased to 150 mm and the muscleculture layer was scraped off for Western analysis as described inSklar, R., and Brown, R. (J. Neurol. Sci. 101:73–81, 1991). The resultspresented in Table A indicate that rhGGF2 treatment increases the levelsof several muscle specific proteins, including dystrophin, myosin heavychain (MHC, adult slow and fast isoforms), but does not increase thelevels of HSP72 or MHC neonate isoform to a similar level per amount ofprotein loaded on the Western. The levels of muscle specific proteinsinduced by rhGGF2 were similar to the quantitative increases in thenumber of myonuclei/mm² (Table 3).

TABLE 3 rhGGF2 Treat- Control ±SD ment ±SD p value Total Protein (μg) 554 ± 38.4  798 ± 73.6 0.007 Myonuclei/mm² 29.0 ± 12.2  106 ± 24.10.008 MHC fast/μg protein 1.22 ± 0.47 4.00 ± 0.40 0.001 MHC slow/μgprotein 0.17 ± 0.13 1.66 ± 0.27 0.001 MHC neonate/μg protein 0.30 ± 0.270.55 ± 0.04 0.199 dystrophin/μg protein 6.67 ± 0.37 25.5 ± 11.0 0.042HSP 72/μg protein 3.30 ± 0.42 4.54 ± 0.08 0.008

The rhGGF2 dependent increase in the adult myosin heavy chain isoforms(slow is found in type I human muscle fibers; fast is found in type 2Aand 2B human muscle fibers) may represent a maturation of the myotubes,as the neonatal isoform was not significantly increased by rhGGF2treatment.

During rat muscle development MHC isoforms switch from fetal to neonatalforms followed by a switch to mature adult slow and fast MHC isoforms(Periasamy et al. J. Biol. Chem. 259:13573–13578, 1984; Periasamy et al.J. Biol. Chem. 260:15856–15862, 1985; Wieczorek et al. J. Cell Biol.101:618–629, 1985). While muscle can autonomously undergo some of theseisoform transitions in the absence of neural cells or tissue, mousemuscle explants express the adult fast MHC isoform only when cultured inthe presence of mouse spinal cord (Ecob-Prince et al. J. Cell Biol.103:995–1005, 1986). Additional evidence that MHC isoform transitionsare influenced by nerve was established by Whalen et al. (Deve. Biol.141:24–40, 1990); after regeneration of notexin treated rat soleusmuscles only the adult fast MHC isoform was produced in the newdenervated muscle, but innervated regenerated muscle made both fast andslow adult MHC isoforms. Thus the demonstration in Table 3 that rhGGF2increases the synthesis of adult MHC isoforms indicates that rhGGF2 mayinduce a developmental maturation of muscle which may mimic neuronalinnervation.

EXAMPLE 7

Neuregulins, Including rhGGF2, Induce the Synthesis of AcetylcholineReceptors in Muscle.

The expression of acetylcholine receptor (AchR) subunit proteins can beinduced by exposing muscle cells to neuregulins. More specifically, wehave shown that contacting muscle cells with rhGGF2 can induce thesynthesis of AchR subunit proteins. This induction following rhGGF2exposure was observed in two ways: first, we detected increasedexpression of human growth hormone via the product of a reporter geneconstruct and second we detected increased binding of alpha-bungarotoxinto cells.

In the following example a mouse myoblast cell line C2 was used. C2cells were transfected with a transgene that contained the 5′ regulatorysequences of the AChR delta subunit gene of mouse linked to a humangrowth hormone full-length cDNA (Baldwin and Burden, 1988. J. Cell Biol.107:2271–2279). This reporter construct allows the measurement of theinduction of AChR delta gene expression by assaying the quantity ofgrowth hormone secreted into the media. The line can be induced to formmyotubes by lowering serum concentration in the media from 20% to 0.5%.

Specifically, mouse C2 myoblasts transfected with an AChR-human growthhormone reporter construct and were assayed for expression of hGHfollowing treatment with rhGGF2. The results of two separate experimentsare summarized in Table 4 and in FIGS. 6 (hGH expression) and 7 (hGHexpression and alpha-bungarotoxin binding). Shown are the dose responsecurves for secreted human growth hormone and for bungarotoxin bindingfrom muscle cultures treated with rhGGF2.

TABLE 4 Effects of rhGGF2 on the expression of AChR delta subunit/hGHtransgene and the synthesis of AChR Exp 1 Exp 2 GGF hGH hGH AChR (ul)(ng/ml) (ng/ml) (cpm/mg protein) 0 9.3 + 2.1 5.7 + 2.1 822 + 170 0.1 —6.8 + 1.5 891 + 134 0.5 — 12.0 + 0.9  993 + 35  1.0 — 9.7 + 2.3 818 +67  5.0 17.5 + 2.8  14.7 + 3.5  1300 + 177  10.0 14.3 + 3.2  14.1 + 3.3 1388 + 137  15.0 22.0 + 1.4  — —

C2 myotubes were treated with cold α-BTX (20 nM) for 1 hr. at 37° C.,washed with culture medium twice and then treated with GGF2. Culturemedium was adjusted with bovine serum albumin at the concentration of 1mg/ml. 24 hours later, culture medium was removed and saved for hGHassay. Muscle cultures were treated with ¹²⁵I-α-BTX (20 nM) for 1 hourat 37° C., washed and scraped in PBS containing 1% SDS. Non-specificbinding was determined in the presence of cold α-BTX (40 nM). The cellhomogenate was counted for radioactivity and assayed for total proteinamount.

The presence of rhGGF2 led to a greater than 2-fold increase in hGH geneexpression, thereby indicating that rhGGF2 induced the synthesis of thedelta subunit of the acetylcholine receptor. Furthermore, increasedbungarotoxin binding is consistent with assembly of these subunitproteins into functional acetylcholine receptors. To strenthen theinterpretation of these data the analysis was repeated on cultures thathad the hGH reporter linked to a metallothiene promotor, which shouldnot be responsive to rhGGF2. The results of that control experimentshowed that the hGH response was mediated through transcriptionalactivation of the AchR delta subunit gene control elements.

These results indicate that rhGGF2 could be useful in replenishing AchRsas part of the therapy for the autoimmune disease Myasthenia gravis.This activity may also be beneficial in treatment of peripheral nerveregeneration and neuropathy by stimulating a key step in re-innervationof muscle.

EXAMPLE 8

Additional Mitogenic Activities of Purified GGF-I and GGF-II

The mitogenic activity of a highly purified sample containing both GGFsI and II was studied using a quantitative method, which allows a singlemicroculture to be examined for DNA synthesis, cell morphology, cellnumber and expression of cell antigens. This technique has been modifiedfrom a method previously reported by Muir et al., AnalyticalBiochemistry 185, 377–382, 1990. The main modifications are: 1) the useof uncoated microtiter plates, 2) the cell number per well, 3) the useof 5% Foetal Bovine Plasma (FBP) instead of 10% Foetal Calf Serum (FCS),and 4) the time of incubation in presence of mitogens andbromodeoxyuridine (BrdU), added simultaneously to the cultures. Inaddition the cell monolayer was not washed before fixation to avoid lossof cells, and the incubation time of monoclonal mouse anti-BrdU antibodyand peroxidase conjugated goat anti-mouse immunoglobulin (IgG) antibodywere doubled to increase the sensitivity of the assay. The assay,optimized for rat sciatic nerve Schwann cells, has also been used forseveral cell lines, after appropriate modifications to the cell cultureconditions.

I. Methods of Mitogenesis Testing

On day 1, purified Schwann cells were plated onto uncoated 96 wellplates in 5% FBP/Dulbecco's Modified Eagle Medium (DMEM) (5,000cells/well). On day 2, GGFs or other test factors were added to thecultures, as well as BrdU at a final concentration of 10 μm. After 48hours (day 4) BrdU incorporation was terminated by aspirating the mediumand cells were fixed with 200 μl/well of 70% ethanol for 20 min at roomtemperature. Next, the cells were washed with water and the DNAdenatured by incubation with 100 μl 2N HCl for 10 min at 37° C.Following aspiration, residual acid was neutralized by filling the wellswith 0.1 M borate buffer, pH 9.0, and the cells were washed withphosphate buffered saline (PBS). Cells were then treated with 50 μl ofblocking buffer (PBS containing 0.1% Triton X 100 and 2% normal goatserum) for 15 min at 37° C. After aspiration, monoclonal mouse anti-BrdUantibody (Dako Corp., Santa Barbara, Calif.) (50 μl/well, 1.4 μg/mldiluted in blocking buffer) was added and incubated for two hours at 37°C. Unbound antibodies were removed by three washes in PBS containing0.1% Triton X-100 and peroxidase-conjugated goat anti-mouse IgG antibody(Dako Corp., Santa Barbara, Calif.) (50 μl/well, 2 μg/ml diluted inblocking buffer) was added and incubated for one hour at 37° C. Afterthree washes in PBS/Triton and a final rinse in PBS, wells received 100μl/well of 50 mM phosphate/citrate buffer, pH 5.0, containing 0.05% ofthe soluble chromogen o-phenylenediamine (OPD) and 0.02% H₂0₂. Thereaction was terminated after 5–20 min at room temperature, by pipetting80 μl from each well to a clean plate containing 40 μl/well of 2Nsulfuric acid. The absorbance was recorded at 490 nm using a platereader (Dynatech Labs). The assay plates containing the cell monolayerswere washed twice with PBS and immunocytochemically stained for BrdU-DNAby adding 100 μl/well of the substrate diaminobenzidine (DAB) and 0.02%H₂0₂ to generate an insoluble product. After 10–20 min the stainingreaction was stopped by washing with water, and BrdU-positive nucleiobserved and counted using an inverted microscope. occasionally,negative nuclei were counterstained with 0.001% Toluidine blue andcounted as before.

II. Cell Lines Used for Mitogenesis Assays

Swiss 3T3 Fibroblasts: Cells, from Flow Labs, were maintained in DMEMsupplemented with 10% FCS, penicillin and streptomycin, at 37° C. in ahumidified atmosphere of 10% C0₂ in air. Cells were fed or subculturedevery two days. For mitogenic assay, cells were plated at a density of5,000 cells/well in complete medium and incubated for a week until cellswere confluent and quiescent. The serum containing medium was removedand the cell monolayer washed twice with serum free-medium. 100 μl ofserum free medium containing mitogens and 10 μM of BrdU were added toeach well and incubated for 48 hours. Dose responses to GGFs and serumor PDGF (as a positive control) were performed.

BHK (Baby Hamster Kidney) 21 C13 Fibroblasts: Cells from EuropeanCollection of Animal Cell Cultures (ECACC), were maintained in GlasgowModified Eagle Medium (GMEM) supplemented with 5% tryptose phosphatebroth, 5% FCS, penicillin and streptomycin, at 37° C. in a humidifiedatmosphere of 5% C0₂ in air. Cells were fed or subcultured every two tothree days. For mitogenic assay, cells were plated at a density of 2,000cell/well in complete medium for 24 hours. The serum containing mediumwas then removed and after washing with serum free medium, replaced with100 μl of 0.1% FCS containing GMEM or GMEM alone. GGFs and FCS or bFGFas positive controls were added, coincident with 10 μM BrdU, andincubated for 48 hours. Cell cultures were then processed as describedfor Schwann cells.

C6 Rat Glioma Cell Line: Cells, obtained at passage 39, were maintainedin DMEM containing 5% FCS, 5% Horse serum (HS), penicillin andstreptomycin, at 37° C. in a humidified atmosphere of 10% C0₂ in air.Cells were fed or subcultured every three days. For mitogenic assay,cells were plated at a density of 2,000 cells/well in complete mediumand incubated for 24 hours. Then medium was replaced with a mixture of1:1 DMEM and F12 medium containing 0.1% FCS, after washing in serum freemedium. Dose responses to GGFs, FCS and AFGF were then performed andcells were processed through the ELISA as previously described for theother cell types.

PC12 (Rat Adrenal Pheochromocytoma Cells): Cells from ECACC, weremaintained in RPMI 1640 supplemented with 10% HS, 5% FCS, penicillin andstreptomycin, in collagen coated flasks, at 37° C. in a humidifiedatmosphere of 5% C0₂ in air. Cells were fed every three days byreplacing 80% of the medium. For mitogenic assay, cells were plated at adensity of 3,000 cells/well in complete medium, on collagen coatedplates (50 μl/well collagen, Vitrogen Collagen Corp., diluted 1:50, 30min at 37° C.) and incubated for 24 hours. The medium was then placedwith fresh RPMI either alone or containing 1 mM insulin or 1% FCS. Doseresponses to FCS/HS (1:2) as positive control and to GGFs were performedas before. After 48 hours cells were fixed and the ELISA performed aspreviously described.

III. Results of Mitogenesis Assays: All the experiments presented inthis Example were performed using a highly purified sample from aSepharose 12 chromatography purification step containing a mixture ofGGF-I and GGF-II (GGFs).

First, the results obtained with the BrdU incorporation assay werecompared with the classical mitogenic assay for Schwann cells based on[125]I-UdR incorporation into DNA of dividing cells, described by J. P.Brockes (Methods Enzymol. 147:217, 1987).

FIG. 12 shows the comparison of data obtained with the two assays,performed in the same cell culture conditions (5,000 cells/well, in 5%FBP/DMEM, incubated in presence of GGFs for 48 hrs). As clearly shown,the results are comparable, but BrdU incorporation assay appears to beslightly more sensitive, as suggested by the shift of the curve to theleft of the graph, i.e. to lower concentrations of GGFS.

As described under the section “Methods of Mitogenesis Testing”, afterthe immunoreactive BrdU-DNA has been quantitated by reading theintensity of the soluble product of the OPD peroxidase reaction, theoriginal assay plates containing cell monolayers can undergo the secondreaction resulting in the insoluble DAB product, which stains the BrdUpositive nuclei. The microcultures can then be examined under aninverted microscope, and cell morphology and the numbers ofBrdU-positive and negative nuclei can be observed.

In FIG. 13A and FIG. 13B the BrdU-DNA immunoreactivity, evaluated byreading absorbance at 490 nm, is compared to the number of BrdU-positivenuclei and to the percentage of BrdU-positive nuclei on the total numberof cells per well, counted in the same cultures. Standard deviationswere less than 10%. The two evaluation methods show a very goodcorrelation and the discrepancy between the values at the highest doseof GGFs can be explained by the different extent of DNA synthesis incells detected as BrdU-positive.

The BrdU incorporation assay can therefore provide additional usefulinformation about the biological activity of polypeptides on Schwanncells when compared to the (125) I-UdR incorporation assay. For example,the data reported in FIG. 15 show that GGFs can act on Schwann cells toinduce DNA synthesis, but at lower doses to increase the number ofnegative cells present in the microculture after 48 hours.

The assay has then been used on several cell lines of different origin.In FIG. 15 the mitogenic responses of Schwann cells and Swiss 3T3fibroblasts to GGFs are compared; despite the weak response obtained in3T3 fibroblasts, some clearly BrdU-positive nuclei were detected inthese cultures. Control cultures were run in parallel in presence ofseveral doses of FCS or human recombinant PDGF, showing that the cellscould respond to appropriate stimuli (not shown).

The ability of fibroblasts to respond to GGFs was further investigatedusing the BHK 21 C13 cell line. These fibroblasts, derived from kidney,do not exhibit contact inhibition or reach a quiescent state whenconfluent. Therefore the experimental conditions were designed to have avery low background proliferation without compromising the cellviability. GGFs have a significant mitogenic activity on BHK21 C13 cellsas shown by FIG. 16 and FIG. 17. FIG. 16 shows the Brdu incorporationinto DNA by BHK 21 C13 cells stimulated by GGFS in the presence of 0.1%FCS. The good mitogenic response to FCS indicates that cell cultureconditions were not limiting. In FIG. 17 the mitogenic effect of GGFs isexpressed as the number of BrdU-positive and BrdU-negative cells and asthe total number of cells counted per well. Data are representative oftwo experiments run in duplicates; at least three fields per well werecounted. As observed for Schwann cells in addition to a proliferativeeffect at low doses, GGFs also increase the numbers of nonrespondingcells surviving. The percentage of BrdU positive cells is proportionalto the increasing amounts of GGFs added to the cultures. The totalnumber of cells after 48 hours in presence of higher doses of GGFs is atleast doubled, confirming that GGFs induce DNA synthesis andproliferation in BHK21 C13 cells. Under the same conditions, cellsmaintained for 48 hours in the presence of 2% FCS showed an increase ofabout six fold (not shown).

C6 glioma cells have provided a useful model to study glial cellproperties. The phenotype expressed seems to be dependent on the cellpassage, the cells more closely resembling an astrocyte phenotype at anearly stage, and an oligodendrocyte phenotype at later stages (beyondpassage 70). C6 cells used in these experiments were from passage 39 topassage 52. C6 cells are a highly proliferating population, thereforethe experimental conditions were optimized to have a very low backgroundof BrdU incorporation. The presence of 0.1% serum was necessary tomaintain cell viability without significantly affecting the mitogenicresponses, as shown by the dose response to FCS (FIG. 18).

In FIG. 19 the mitogenic responses to aFGF (acidic Fibroblast growthfactor) and GGFs are expressed as the percentages of maximal BrdUincorporation obtained in the presence of FCS (8%). Values are averagesof two experiments, run in duplicates. The effect of GGFs was comparableto that of a pure preparation of aFGF. aFGF has been described as aspecific growth factor for C6 cells (Lim R. et al., Cell Regulation1:741–746, 1990) and for that reason it was used as a positive control.The direct counting of BrdU positive and negative cells was not possiblebecause of the high cell density in the microcultures. In contrast tothe cell lines so far reported, PC12 cells did not show any evidentresponsiveness to GGFS, when treated under culture conditions in whichPC12 could respond to sera (mixture of FCS and HS as used routinely forcell maintenance). Nevertheless the number of cells plated per wellseems to affect the behavior of PC12 cells, and therefore furtherexperiments are required.

EXAMPLE 9 Amino Acid Sequences of Purified GGF-I and GGF-II

Amino acid sequence analysis studies were performed using highlypurified bovine pituitary GGF-I and GGF-II. The conventional singleletter code was used to describe the sequences. Peptides were obtainedby lysyl endopeptidase and protease V8 digests, carried out on reducedand carboxymethylated samples, with the lysyl endopeptidase digest ofGGF-II carried out on material eluted from the 55–65 RD region of a 11%SDS-PAGE (MW relative to the above-quoted markers).

A total of 21 peptide sequences (see FIG. 8, SEQ ID Nos. 1–20, 169) wereobtained for GGF-I, of which 12 peptides (see FIG. 9, SEQ ID Nos. 1,22–29, 17, 19, and 32) are not present in current protein databases andtherefore represent unique sequences. A total of 12 peptide sequences(see FIG. 10, (SEQ ID Nos. 33–39, 51–52) and 160–162) were obtained forGGF-II, of which 10 peptides (see FIG. 11, SEQ ID NOS: 42–50) are notpresent in current protein databases and therefore represent uniquesequences (an exception is peptide GGF-II 06 which shows identicalsequences in many proteins which are probably of no significance giventhe small number of residues). These novel sequences are extremelylikely to correspond to portions of the true amino acid sequences ofGGFs I and II.

Particular attention can be drawn to the sequences of GGF-I 07 andGGF-II 12, which are clearly highly related. The similarities indicatethat the sequences of these peptides are almost certainly those of theassigned GGF species, and are most unlikely to be derived fromcontaminant proteins.

In addition, in peptide GGF-II 02, the sequence X S S is consistent withthe presence of an N linked carbohydrate moiety on an asparagine at theposition denoted by X.

In general, in FIGS. 8 and 10, X represents an unknown residue denotinga sequencing cycle where a single position could not be called withcertainty either because there was more than one signal of equal size inthe cycle or because no signal was present. As asterisk denotes thosepeptides where the last amino acid called corresponds to the last aminoacid present in that peptide. In the remaining peptides, the signalstrength after the last amino acid called was insufficient to continuesequence calling to the end of that peptide. The right hand columnindicates the results of a computer database search using the GCGpackage FASTA and TFASTA programs to analyze the NBRF and EMBL sequencedatabases. The name of a protein in this column denotes identity of aportion of its sequence with the peptide amino acid sequence calledallowing a maximum of two mismatches. A question mark denotes threemismatches allowed. The abbreviations used are as follows:

-   HMG-1 High Mobility Group protein-1-   HMG-2 High Mobility Group protein-2-   LH-alpha Luteinizing hormone alpha subunit-   LH-beta Luteinizing hormone beta subunit

EXAMPLE 10

Isolating and Cloning of Nucleotide Sequences Encoding ProteinsContaining GGF-I and GGF-II Peptides

Isolation and cloning of the GGF-II nucleotide sequences was performedas outlined herein, using peptide sequence information and libraryscreening, and was performed as set out below. It will be appreciatedthat the peptides of FIGS. 10 and 11 can be used as the starting pointfor isolation and cloning of GGF-I sequences by following the techniquesdescribed herein. Indeed, FIG. 20, SEQ ID Nos. 54–76, 78–88 showspossible degenerate oligonucleotide probes for this purpose, and FIG.22, SEQ ID NOS: 56–115, lists possible PCR primers. DNA sequence andpolypeptide sequence should be obtainable by this means as with GGF-II,and also DNA constructs and expression vectors incorporating such DNAsequence, host cells genetically altered by incorporating suchconstructs/vectors, and protein obtainable by cultivating such hostcells. The invention envisages such subject matter.

I. Design and Synthesis of Oligonucleotide Probes and Primers

Degenerate DNA oligomer probes were designed by backtranslating theamino acid sequences (derived from the peptides generated from purifiedGGF protein) into nucleotide sequences. Oligomers represented either thecoding strand or the non-coding strand of the DNA sequence. When serine,arginine or leucine were included in the oligomer design, then twoseparate syntheses were prepared to avoid ambiguities. For example,serine was encoded by either TCN or AGY as in 537 and 538 or 609 and610. Similar codon splitting was done for arginine or leucine (e.g. 544,545). DNA oligomers were synthesized on a Biosearch 8750 4-column DNAsynthesizer using β-cyanoethyl chemistry operated at 0.2 micromole scalesynthesis. Oligomers were cleaved off the column (500 angstrom CpGresins) and deprotected in concentrated ammonium hydroxide for 6–24hours at 55–60° C. Deprotected oligomers were dried under vacuum(Speedvac) and purified by electrophoresis in gels of 15% acrylamide (20mono: 1 bis), 50 mM Tris-borate-EDTA buffer containing 7M urea. Fulllength oligomers were detected in the gels by UV shadowing, then thebands were excised and DNA oligomers eluted into 1.5 mls H20 for 4–16hours with shaking. The eluate was dried, redissolved in 0.1 ml H₂0 andabsorbance measurements were taken at 260 nm.

Concentrations were determined according to the following formula:(A 260×units/ml) (60.6/length=ΔμM)

All oligomers were adjusted to 50 μM concentration by addition of H₂0.

Degenerate probes designed as above are shown in FIG. 20, SEQ ID Nos.54–76, 78–88.

PCR primers were prepared by essentially the same procedures that wereused for probes with the following modifications. Linkers of thirteennucleotides containing restriction sites were included at the 5′ ends ofthe degenerate oligomers for use in cloning into vectors. DNA synthesiswas performed at 1 micromole scale using 1,000 angstrom CpG resins andinosine was used at positions where all four nucleotides wereincorporated normally into degenerate probes. Purifications of PCRprimers included an ethanol precipitation following the gelelectrophoresis purification.

II. Library Construction and Screening

A bovine genomic DNA library was purchased from Stratagene (CatalogueNumber: 945701). The library contained 2×10⁶ 15–20 kb Sau3Al partialbovine DNA fragments cloned into the vector lambda DashII. A bovinetotal brain cDNA library was purchased from Clonetech (Catalogue Number:BL 10139). Complementary DNA libraries were constructed (In Vitrogen;Stratagene) from mRNA prepared from bovine total brain, from bovinepituitary and from bovine posterior pituitary. In Vitrogen prepared twocDNA libraries: one library was in the vector lambda g10, the other invector pcDNAI (a plasmid library). The Stratagene libraries wereprepared in the vector lambda unizap. Collectively, the cDNA librariescontained 14 million primary recombinant phage.

The bovine genomic library was plated on E. coli K12 host strain LE392on 23×23 cm plates (Nunc) at 150,000 to 200,000 phage plaques per plate.Each plate represented approximately one bovine genome equivalent.Following an overnight incubation at 37° C., the plates were chilled andreplicate filters were prepared according to procedures of Maniatis etal. (2:60–81). Four plaque lifts were prepared from each plate ontouncharged nylon membranes (Pall Biodyne A or MSI Nitropure). The DNA wasimmobilized onto the membranes by cross-linking under UV light for 5minutes or, by baking at 80° C. under vacuum for two hours. DNA probeswere labelled using T4 polynucleotide kinase (New England Biolabs) withgamma 32P ATP (New England Nuclear; 6500 Ci/mmol) according to thespecifications of the suppliers. Briefly, 50 pmols of degenerate DNAoligomer were incubated in the presence of 600 μCi gamma ³²P-ATP and 5units T4 polynucleotide kinase for 30 minutes at 37° C. Reactions wereterminated, gel electrophoresis loading buffer was added and thenradiolabelled probes were purified by electrophoresis. 32P labelledprobes were excised from gel slices and eluted into water.Alternatively, DNA probes were labelled via PCR amplification byincorporation of α-32P-dATP or α-32P dCTP according to the protocol ofSchowalter and Sommer, Anal. Biochem 177:90–94 (1989). Probes labelledin PCR reactions were purified by desalting on Sephadex G-150 columns.

Prehybridization and hybridization were performed in GMC buffer (0.52 MNaPi, 7% SDS, 1% BSA, 1.5 mM EDTA, 0.1 M NaCl 10 mg/ml tRNA). Washingwas performed in oligowash (160 ml 1 M Na₂HPO₄, 200 ml 20% SDS, 8.0 ml0.5 M EDTA, 100 ml 5M NaCl, 3632 ml H20). Typically, 20 filters (400 sq.centimeters each) representing replicate copies of ten bovine genomeequivalents were incubated in 200 ml hybridization solution with 100pmols of degenerate oligonucleotide probe (128–512 fold degenerate).Hybridization was allowed to occur overnight at 5° C. below the minimummelting temperature calculated for the degenerate probe. The calculationof minimum melting temperature assumes 2° C. for an AT pair and 4° C.for a GC pair.

Filters were washed in repeated changes of oligowash at thehybridization temperatures four to five hours and finally, in 3.2Mtetramethylammonium chloride, 1% SDS twice for 30 min at a temperaturedependent on the DNA probe length. For 20mers, the final washtemperature was 60° C. Filters were mounted, then exposed to X-ray film(Kodak XAR5) using intensifying screens (Dupont Cronex Lightening Plus).Usually, a three to five day film exposure at minus 80° C. wassufficient to detect duplicate signals in these library screens.Following analysis of the results, filters could be stripped andreprobed. Filters were stripped by incubating through two successivecycles of fifteen minutes in a microwave oven at full power in asolution of 1% SDS containing 10 mM EDTA pH8. Filters were taken throughat least three to four cycles of stripping and reprobing with variousprobes.

III. Recombinant Phage Isolation, Growth and DNA Preparation

These procedures followed standard protocol as described in RecombinantDNA (Maniatis et al 2:60–2:81).

IV. Analysis of Isolated Clones Using DNA Digestion and Southern Blots

Recombinant Phage DNA samples (2 micrograms) were digested according toconditions recommended by the restriction endonuclease supplier (NewEngland Biolabs). Following a four hour incubation at 37° C., thereactions products were precipitated in the presence of 0.1M sodiumacetate and three volumes of ethanol. Precipitated DNA was collected bycentrifugation, rinsed in 75% ethanol and dried. All resuspended sampleswere loaded onto agarose gels (typically 1% in TAE buffer; 0.04M Trisacetate, 0.002M EDTA). Gel runs were at 1 volt per centimeter from 4 to20 hours. Markers included lambda Hind III DNA fragments and/orØX174HaeIII DNA fragments (New England Biolabs). The gels were stainedwith 0.5 micrograms/ml of ethidium bromide and photographed. Forsouthern blotting, DNA was first depurinated in the gel by treatmentwith 0.125 N HCl, denatured in 0.5 N NaOH and transferred in 20×SSC (3Msodium chloride, 0.03 M sodium citrate) to uncharged nylon membranes.Blotting was done for 6 hours up to 24 hours, then the filters wereneutralized in 0.5 Tris HCl pH 7.5, 0.15 M sodium chloride, then rinsedbriefly in 50 mM Tris-borate EDTA.

For cross-linking, the filters were wrapped first in transparent plasticwrap, then the DNA side exposed for five minutes to an ultravioletlight. Hybridization and washing was performed as described for libraryscreening (see section 2 of this Example). For hybridization analysis todetermine whether similar genes exist in other species slightmodifications were made. The DNA filter was purchased from Clonetech(Catalogue Number 7753-1) and contains 5 micrograms of EcoRI digestedDNA from various species per lane. The probe was labelled by PCRamplification reactions as described in section 2 above, andhybridizations were done in 80% buffer B(2 g polyvinylpyrrolidine, 2 gFicoll-400, 2 g bovine serum albumin, 50 ml 1M Tris-HCl (pH 7.5) 58 gNaCl, 1 g sodium pyrophosphate, 10 g sodium dodecyl sulfate, 950 ml H₂0)containing 10% dextran sulfate. The probes were denatured by boiling forten minutes then rapidly cooling in ice water. The probe was added tothe hybridization buffer at 10⁶ dpm ³²P per ml and incubated overnightat 60° C. The filters were washed at 60° C. first in buffer B followedby 2×SSC, 0.1% SDS then in 1×SSC, 0.1% SDS. For high stringency,experiments, final washes were done in 0.1×SSC, 1% SDS and thetemperature raised to 65° C.

Southern blot data were used to prepare a restriction map of the genomicclone and to indicate which subfragments hybridized to the GGF probes(candidates for subcloning).

V. Subcloning of Segments of DNA Homologous to Hybridization Probes

DNA digests (e.g. 5 micrograms) were loaded onto 1% agarose gels thenappropriate fragments excised from the gels following staining. The DNAwas purified by adsorption onto glass beads followed by elution usingthe protocol described by the supplier (Bio 101). Recovered DNAfragments (100–200 ng) were ligated into linearized dephosphorylatedvectors, e.g. pT3T7 (Ambion), which is a derivative of pUC18, using T4ligase (New England Biolabs). This vector carries the E. coli βlactamase gene, hence, transformants can be selected on platescontaining ampicillin. The vector also supplies β-galactosidasecomplementation to the host cell, therefore non-recombinants (blue) canbe detected using isopropylthiogalactoside and Bluogal (BethesdaResearch Labs). A portion of the ligation reactions was used totransform E. coli K12 XL1 blue competent cells (Stratagene CatalogueNumber: 200236) and then the transformants were selected on LB platescontaining 50 micrograms per ml ampicillin. White colonies were selectedand plasmid mini preps were prepared for DNA digestion and for DNAsequence analysis. Selected clones were retested to determine if theirinsert DNA hybridized with the GGF probes.

VI. DNA Sequencing

Double stranded plasmid DNA templates were prepared from 5 ml culturesaccording to standard protocols. Sequencing was by the dideoxy chaintermination method using Sequenase 2.0 and a dideoxynucleotidesequencing kit (US Biochemical) according to the manufacturers protocol(a modification of Sanger et al. PNAS; USA 74:5463 (1977)].Alternatively, sequencing was done in a DNA thermal cycler (PerkinElmer, model 4800) using a cycle sequencing kit (New England Biolabs;Bethesda Research Laboratories) and was performed according tomanufacturers instructions using a 5′-end labelled primer. Sequenceprimers were either those supplied with the sequencing kits or weresynthesized according to sequence determined from the clones. Sequencingreactions were loaded on and resolved on 0.4 mm thick sequencing gels of6% polyacrylamide. Gels were dried and exposed to X-Ray film. Typically,35S was incorporated when standard sequencing kits were used and a 32Pend labelled primer was used for cycle sequencing reactions. Sequenceswere read into a DNA sequence editor from the bottom of the gel to thetop (5′ direction to 3′) and data were analyzed using programs suppliedby Genetics Computer Group (GCG, University of Wisconsin).

VII. RNA Preparation and PCR Amplification

Open reading frames detected in the genomic DNA and which containedsequence encoding GGF peptides were extended via PCR amplification ofpituitary RNA. RNA was prepared from frozen bovine tissue (Pelfreeze)according to the guanidine neutral-CsCl procedure (Chirgwin et. al.Biochemistry 18:5294(1979).) Polyadenylated RNA was selected by oligo-dTcellulose column chromatography (Aviv and Leder PNAS (USA) 69:1408(1972)).

Specific DNA target sequences were amplified beginning with either totalRNA or polyadenylated RNA samples that had been converted to cDNA usingthe Perkin Elmer PCR/RNA Kit Number: N808-0017. First strand reversetranscription reactions used 1 μg template RNA and either primers ofoligo dT with restriction enzyme recognition site linkers attached orspecific antisense primers determined from cloned sequences withrestriction sites attached. To produce the second strand, the primerseither were plus strand unique sequences as used in 3′ RACE reactions(Frohman et. al., PNAS (USA) 85:8998 (1988)) or were oligo dT primerswith restriction sites attached if the second target site had been addedby terminal transferase tailing first strand reaction products with dATP(e.g. 5′ race reactions, Frohman et. al., ibid). Alternatively, as inanchored PCR reactions the second strand primers were degenerate, hence,representing particular peptide sequences.

The amplification profiles followed the following general scheme: 1)five minutes soak file at 95° C.; 2) thermal cycle file of 1 minute, 95°C.; 1 minute ramped down to an annealing temperature of 45° C., 50° C.or 55° C.; maintain the annealing temperature for one minute; ramp up to72° C. over one minute; extend at 72° C. for one minute or for oneminute plus a 10 second auto extension; 3) extension cycle at 72° C.,five minutes, and; 4) soak file 4° C. for infinite time. Thermal cyclefiles (#2) usually were run for 30 cycles. A sixteen μl sample of each100 μl amplification reaction was analyzed by electrophoresis in 2%Nusieve 1% agarose gels run in TAE buffer at 4 volts per centimeter forthree hours. The gels were stained, then blotted to uncharged nylonmembranes which were probed with labelled DNA probes that were internalto the primers.

Specific sets of DNA amplification products could be identified in theblotting experiments and their positions used as a guide to purificationand reamplification. When appropriate, the remaining portions ofselected samples were loaded onto preparative gels, then followingelectrophoresis four to five slices of 0.5 mm thickness (bracketing theexpected position of the specific product) were taken from the gel. Theagarose was crushed, then soaked in 0.5 ml of electrophoresis bufferfrom 2–16 hours at 40° C. The crushed agarose was centrifuged for twominutes and the aqueous phase was transferred to fresh tubes.

Reamplification was done on five microliters (roughly 1% of the product)of the eluted material using the same sets of primers and the reactionprofiles as in the original reactions. When the reamplificationreactions were completed, samples were extracted with chloroform andtransferred to fresh tubes. Concentrated restriction enzyme buffers andenzymes were added to the reactions in order to cleave at therestriction sites present in the linkers. The digested PCR products werepurified by gel electrophoresis, then subcloned into vectors asdescribed in the subcloning section above. DNA sequencing was donedescribed as above.

VIII. DNA Sequence Analysis

DNA sequences were assembled using a fragment assembly program and theamino acid sequences deduced by the GCG programs GelAssemble, Map andTranslate. The deduced protein sequences were used as a query sequenceto search protein sequence databases using WordSearch. Analysis was doneon a VAX Station 3100 workstation operating under VMS 5.1. The databasesearch was done on SwissProt release number 21 using GCG Version 7.0.

IX. Results of Cloning and Sequencing of Genes Encoding GGF-I and GGF-II

As indicated above, to identify the DNA sequence encoding bovine GGF-IIdegenerate oligonucleotide probes were designed from GGF-II peptidesequences. GGF-II 12 (SEQ ID No: 52), a peptide generated via lysylendopeptidase digestion of a purified GGF-II preparation (see FIGS. 16and 12) showed strong amino acid sequence homology with GGF-I 07 (SEQ IDNo. 24), a tryptic peptide generated from a purified GGF-I preparation.GGF-II 12 was thus used to create ten degenerate oligonucleotide probes(see oligos 609, 610 and 649 to 656 in FIG. 20, SEQ ID NOS: 66, 67, 68and 75 71–79 respectively, respectively). A duplicate set of filterswere probed with two sets (set 1=609, 610; set 2=649–5656) of probesencoding two overlapping portions of GGF-II 12. Hybridization signalswere observed, but, only one clone hybridized to both probe sets. Theclone (designated GGF2BG1) was purified.

Southern blot analysis of DNA from the phage clone GGF2BG1 confirmedthat both sets of probes hybridized with that bovine DNA sequence, andshowed further that both probes reacted with the same set of DNAfragments within the clone. Based on those experiments a 4 kb Eco RIsub-fragment of the original clone was identified, subcloned andpartially sequenced. FIG. 21 shows the nucleotide sequence, SEQ ID No.89) and the deduced amino acid sequence of the initial DNA sequencereadings that included the hybridization sites of probes 609 and 650,and confirmed that a port of this bovine genomic DNA encoded peptide 12(KASLADSGEYM) (SEQ ID NO. 129).

Further sequence analysis demonstrated that GGF-II 12 resided on a 66amino acid open reading frame (see below) which has become the startingpoint for the isolation of overlapping sequences representing a putativebovine GGF-II gene and a cDNA.

Several PCR procedures were used to obtain additional coding sequencesfor the putative bovine GGF-II gene. Total RNA and oligo dT-selected(poly A containing) RNA samples were prepared from bovine totalpituitary, anterior pituitary, posterior pituitary, and hypothalamus.Using primers from the list shown in FIG. 22, SEQ ID Nos. 105–115one-sided PCR reactions (RACE) were used to amplify cDNA ends in boththe 3′ and 5′ directions, and anchored PCR reactions were performed withdegenerate oligonucleotide primers representing additional GGF-IIpeptides. FIG. 29 summarizes the contiguous DNA structures and sequencesobtained in those experiments. From the 3′ RACE reactions, threealternatively spliced cDNA sequences were produced, which have beencloned and sequenced. A 5′ RACE reaction led to the discovery of anadditional exon containing coding sequence for at least 52 amino acids.Analysis of that deduced amino acid sequence revealed peptides GGF-II-6and a sequence similar to GGF-I-18 (see below). The anchored PCRreactions led to the identification of (cDNA) coding sequences ofpeptides GGF-II-1, 2, 3 and 10 contained within an additional cDNAsegment of 300 bp. The 5′ limit of this segment (i.e., segment E, seeFIG. 30) is defined by the oligonucleotide which encodes peptideGGF-II-1 and which was used in the PCR reaction (additional 5′ sequencedata exists as described for the human clone in Example 11). Thus thisclone contains nucleotide sequences encoding six out of the existingtotal of nine novel GGF-II peptide sequences.

The cloned gene was characterized first by constructing a physical mapof GGF2BG1 that allowed us to position the coding sequences as they werefound (see below, FIG. 30). DNA probes from the coding sequencesdescribed above have been used to identify further DNA fragmentscontaining the exons on this phage clone and to identify clones thatoverlap in both directions. The putative bovine GGF-II gene is dividedinto at least 5 coding segments. Coding segments are defined as discretelengths of DNA sequence which can be translated into polypeptidesequences using the universal genetic code. The coding segmentsdescribed in FIG. 36 and referred to in the present application are: 1)particular exons present within the GGF gene (e.g. coding segment a), or2) derived from sets of two or more exons that appear in specificsub-groups of mRNAs, where each set can be translated into the specificpolypeptide segments as in the gene products shown. The polypeptidesegments referred to in the claims are the translation products of theanalogous DNA coding segments. Only coding segments A and B have beendefined as exons and sequenced and mapped thus far. The summary of thecontiguous coding sequences identified is shown in FIGS. 31(A–B). Theexons are listed (alphabetically) in the order of their discovery. It isapparent from the intron/exon boundaries that exon B may be included incDNAs that connect coding segment E and coding segment A. That is, exonB cannot be spliced out without compromising the reading frame.Therefore, we suggest that three alternative splicing patterns canproduce putative bovine GGF-II cDNA sequences 1, 2 and 3. The codingsequences of these, designated GGF2BPP1.CDS, GGF2BPP2.CDS andGGF2BPP3.CDS, respectively, are given in FIGS. 27A (SEQ ID NO: 129),27(B–C), (SEQ ID No: 134) and 27(D–E), (SEQ ID No: 135), respectively.The deduced amino acid sequence of the three cDNAs is also given inFIGS. 27A–B(SEQ ID No: 384), 27(B–C), (SEQ ID No: 385) and 27(D–E), (SEQID No: 387).

The three deduced structures encode proteins of lengths 206, 281 and 257amino acids. The first 183 residues of the deduced protein sequence areidentical in all three gene products. At position 184 the clones differsignificantly. A codon for glycine GGT in GGF2BPP1 also serves as asplice donor for GGF2BPP2 and GGF2BPP3, which alternatively add on exonsC, C/D, C/D′ and D or C, C/D and D, respectively, and shown in FIGS.32(A–B), SEQ ID NO: 145). GGFIIBPP1 is a truncated gene product which isgenerated by reading past the coding segment A splice junction into thefollowing intervening sequence (intron). This represents coding segmentA′ in FIG. 30E (SEQ ID NO: 136). The transcript ends adjacent to acanonical AATAAA (SEQ ID NO:420) polyadenylation sequence, and wesuggest that this truncated gene product represents a bona fide maturetranscript. The other two longer gene products share the same 3′untranslated sequence and polyadenylation site.

All three of these molecules contain six of the nine novel GGF-IIpeptide sequences (see FIG. 11) and another peptide is highly homologousto GGF-I-18 (see FIG. 26). This finding gives a high probability thatthis recombinant molecule encodes at least a portion of bovine GGF-II.Furthermore, the calculated isoelectric points for the three peptidesare consistent with the physical properties of GGF-I and II. Since themolecular size of GGF-II is roughly 60 kD, the longest of the threecDNAs should encode a protein with nearly one-half of the predictednumber of amino acids.

A probe encompassing the B and A exons was labelled via PCRamplification and used to screen a cDNA library made from RNA isolatedfrom bovine posterior pituitary. One clone (GGF2BPP5) showed the patternindicated in FIG. 29 and contained an additional DNA coding segment (G)between coding segments A and C. The entire nucleic acid sequence isshown in FIG. 31 (SEQ ID NO: 144). The predicted translation productfrom the longest open reading frame is 241 amino acids. A portion of asecond cDNA (GGF2BPP4) was also isolated from the bovine posteriorpituitary library using the probe described above. This clone showed thepattern indicated in FIG. 29. This clone is incomplete at the 5′ end,but is a splicing variant in the sense that it lacks coding segments Gand D. BPP4 also displays a novel 3′ end with regions H, K and L beyondregion C/D. The sequence of BPP4 is shown in FIG. 33 (SEQ ID NO: 146).

EXAMPLE 11

GGF Sequences in Various Species

The GGF proteins are the members of a new superfamily of proteins. Inhigh stringency cross hybridization studies (DNA blotting experiments)with other mammalian DNAs we have shown, clearly, that DNA probes fromthis bovine recombinant molecule can readily detect specific sequencesin a variety of samples tested. A highly homologous sequence is alsodetected in human genomic DNA. The autoradiogram is shown in FIG. 28.The signals in the lanes containing rat and human DNA represent the ratand human equivalents of the GGF gene, the sequences of several cDNA'sencoded by this gene have been recently reported by Holmes et al.(Science 256: 1205 (1992)) and Wen et al. (Cell 69: 559 (1992)).

EXAMPLE 12

Isolation of a Human Sequence Encoding Human GGF2

Several human clones containing sequences from the bovine GGFII codingsegment E were isolated by screening a human cDNA library prepared frombrain stem (Stratagene catalog #935206). This strategy was pursued basedon the strong link between most of the GGF2 peptides (unique to GGF2)and the predicted peptide sequence from clones containing the bovine Esegment. This library was screened as described in Example 8, Section IIusing the oligonucleotide probes 914–919 listed below.

-   914TCGGGCTCCATGAAGAAGATGTA (SEQ ID NO: 179)-   915TCCATGAAGAAGATGTACCTGCT (SEQ ID NO: 180)-   916ATGTACCTGCTGTCCTCCTTGA (SEQ ID NO: 181)-   917TTGAAGAAGGACTCGCTGCTCA (SEQ ID NO: 182)-   918AAAGCCGGGGGCTTGAAGAA (SEQ ID NO: 183)-   919ATGARGTGTGGGCGGCGAAA (SEQ ID NO: 184)

Clones detected with these probes were further analyzed byhybridization. A probe derived from coding segment A (see FIG. 30),which was produced by labeling a polymerase chain reaction (PCR) productfrom segment A, was also used to screen the primary library. Severalclones that hybridized with both A and E derived probes were selectedand one particular clone, GGF2HBS5, was selected for further analysis.This clone is represented by the pattern of coding segments (EBACC/D′Das shown in FIG. 30). The E segment in this clone is the humanequivalent of the truncated bovine version of E shown in FIG. 30.GGF2HBS5 is the most likely candidate to encode GGF-II of all the“putative” GGF-II candidates described. The length of coding sequencesegment E is 786 nucleotides plus 264 bases of untranslated sequence.The predicted size of the protein encoded by GGF2HBS5 is approximately423 amino acids (approximately 45 kilodaltons, see FIG. 44, SEQ ID NO:170), which is similar to the size of the deglycosylated form of GGF-II(see Example 19). Additionally, seven of the GGF-II peptides listed inFIG. 26 have equivalent sequences which fall within the protein sequencepredicted from region E. Peptides II-6 and II-12 are exceptions, whichfall in coding segment B and coding segment A, respectively. RNAencoding the GGF2HBS5 protein was produced in an in vitro transcriptionsystem driven by the bacteriophage T7 promoter resident in the vector(Bluescript SK [Stratagene Inc.] see FIG. 47) containing the GGF2HBS5insert. This RNA was translated in a cell free (rabbit reticulocyte)translation system and the size of the protein product was 45 Kd.Additionally, the cell-free product has been assayed in a Schwann cellmitogenic assay to confirm biological activity. Schwann cells treatedwith conditioned medium show both increased proliferation as measured byincorporation of ¹²⁵I-Uridine and phosphorylation on tyrosine of aprotein in the 185 kilodalton range.

Thus the size of the product encoded by GGF2HBS5 and the presence of DNAsequences which encode human peptides highly homologous to the bovinepeptides shown in FIG. 11 confirm that GGF2HBS5 encodes the humanequivalent of bovine GGF2. The fact that conditioned media prepared fromcells transformed with this clone elicits Schwann cell mitogenicactivity confirms that the GGFIIHBS5 gene produce (unlike the BPP5 geneproduct) is secreted. Additionally the GGFIIBPP5 gene product seems tomediate the Schwann cell proliferation response via a receptor tyrosinekinase such as p185^(erbB2) or a closely related receptor (see Example18).

EXAMPLE 13

Expression of Human Recombinant GGF2 in Mammalian and Insect Cells

The GGF2HBS5 cDNA clone encoding human GGF2 (as described in Example 12and also referred to herein as HBS5) was cloned into vector pcDL-SRα296and COS-7 cells were transfected in 100 mm dishes by the DEAE-dextranmethod. Cell lysates or conditioned media from transiently expressingCOS cells were harvested at 3 or 4 days post-transfection. To preparelysates, cell monolayers were washed with PBS, scraped from the disheslysed by three freeze/thaw cycles in 150 μm of 0.25 M Tris-HCl, pH8.Cell debris was pelleted and the supernatant recovered. Conditionedmedia samples (7 mls.) were collected, then concentrated and bufferexchanged with 10 mm Tris, pH 7.4 using Centiprep-10 and Centricon-10units as described by the manufactures (Amicon, Beverly, Mass.). Ratnerve Schwann cells were assayed for incorporation of DNA synthesisprecursors, as described. Conditioned media or cell lysate samples weretested in the Schwann cell proliferation assay as described inMarchionni et al., Nature 362:313 (1993). The cDNA, GGF2HBS5, encodingGGF2 directed the secretion of the protein product to the medium.Minimal activity was detectable inside the cells as determined by assaysusing cell lysates. GGF2HFB1 and GGFBPP5 cDNA's failed to direct thesecretion of the product to the extracellular medium. GGF activity fromthese clones was detectable only in cell lysates.

Recombinant GGF2 was also expressed in CHO cells. The GGF2HBS5 cDNAencoding GGF2 was cloned into the EcoRI site of vector pcdhfrpolyA andtransfected into the DHFR negative CHO cell line (GG44) by the calciumphosphate coprecipitation method. Clones were selected in nucleotide andnucleoside free α medium (Gibco) in 96-well plates. After 3 weeks,conditioned media samples from individual clones were screened forexpression of GGF by the Schwann cell proliferation assay as describedin Marchionni et al., Nature 362:313 (1993). Stable clones whichsecreted significant levels of GGF activity into the medium wereidentified. Schwann cell proliferation activity data from differentvolume aliquots of CHO cell conditioned medium were used to produce thedose response curve shown in FIG. 46 (Graham and Van Der Eb, Virology52:456, 1973). This material was analyzed on a Western blot probed withpolyclonal antisera raised against a GGF2 specific peptide. A band ofapproximately 65 Kd (the expected size of GGF2 extracted from pituitary)is specifically labeled (FIG. 48, lane 12).

Recombinant GGF2 was also expressed in insect cells using theBaculovirus expression. Sf9 insect cells were infected with baculoviruscontaining the GGF2HBS5 cDNA clone at a multiplicity of 3–5 (10⁶cells/ml) and cultured in Sf900-II medium. Schwann cell mitogenicactivity was secreted into the extracellular medium. Different volumesof insect cell conditioned medium were tested in the Schwann cellproliferation assay in the absence of forskolin and the data used toproduce a dose response curve.

This material was also analyzed on a Western blot (FIG. 45B) probed withthe GGF II specific antibody described above.

The methods used in this example were as follows:

Schwann cell mitogenic activity of recombinant human and bovine glialgrowth factors was determined as follows: Mitogenic responses ofcultured Schwann cells were measured in the presence of 5 μM forskolinusing crude recombinant GGF preparations obtained from transientmammalian expression experiments. Incorporation of [¹²⁵I]-Urd wasdetermined following an 18-24 hour exposure to materials obtained fromtransfected or mock transfected cos cells as described in the Methods.The mean and standard deviation of four sets of data are shown. Themitogenic response to partially purified native bovine pituitary GGF(carboxymethyl cellulose fraction; Goodearl et al., submitted) is shown(GGF) as a standard of one hundred percent activity.

cDNAs (FIG. 46, SEQ ID NOs. 166–168) were cloned into pcDL-SRα296(Takebe et al., Mol. Cell Biol. 8:466–472 (1988)), and COS-7 cells weretransfected in 100 mm dishes by the DEAE-dextran method (Sambrook etal., In Molecular Cloning. A Laboratory Manual, 2nd. ed. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)). Cell lysatesor conditioned media were harvested at 3 or 4 days post-transfection. Toprepare lysates, cell monolayers were washed with PBS, scraped from thedishes, and lysed by three freeze/than cycles in 150 μl of 0.25 MTris-HCl, pH 8. Cell debris was pelleted and the supernate recovered.Conditioned media samples (7 mls) were collected, then concentrated andbuffer exchanged with 10 mM Tris, pH 7.4 using Centriprep-10 andCentricon-10 units are described by the manufacturers (Amicon, Beverly,Mass.). Rat sciatic nerve Schwann cells were assayed for incorporationof DNA synthesis precursors, as described (Davis and Stroobant, J. CellBiol. 110:1353–1360 (1990); Brockes et al., Brain Res. 165:105–118(1979)).

Western blot of recombinant CHO cell conditioned medium were performedas follows: A recombinant CHO clone was cultured in MCDB302 protein-freefor 3 days. 2 ml of conditioned medium was harvested, concentrated,buffered exchanged against 10 mM Tris-HCl, pH 7.4 and lyophilized todryness. The pellet was resuspended in SDS-PAGE sample buffer, subjectedto reducing SDS gel electrophoresis and analyzed by Western blottingwith a GGF peptide antibody. A CHO control was done by using conditionedmedium from untransfected CHO-DG44 host and the CHO HBS5 levels wereassayed using conditioned medium from a recombinant clone.

EXAMPLE 14

Identification of Functional Elements of GGF

The deduced structures of the family of GGF sequences indicate that thelongest forms (as represented by GGF2BPP4) encode transmembrane proteinswhere the extracellular part contains a domain which resembles epidermalgrowth factor (see Carpenter and Wahl in Peptide Growth Factors andTheir Receptors I pp. 69–133, Springer-Verlag, N.Y. 1991). The positionsof the cysteine residues in coding segments C and C/D or C/D′ peptidesequence are conserved with respect to the analogous residues in theepidermal growth factor (EGF) peptide sequence (see FIG. 34, SEQ ID NOS:147–149). This suggests that the extracellular domain functions asreceptor recognition and biological activation sites. Several of thevariant forms lack the H, K, and L coding segments and thus may beexpressed as secreted, diffusible biologically active proteins. GGF DNAsequences encoding polypeptides which encompass the EGF-like domain(EGFL) can have full biological activity for stimulating glial cellmitogenic activity.

Membrane bound versions of this protein may induce Schwann cellproliferation if expressed on the surface of neurons duringembryogenesis or during nerve regeneration (where the surfaces ofneurons are intimately associated with the surfaces of proliferatingSchwann cells).

Secreted (non membrane bound) GGFs may act as classically diffusiblefactors which can interact with Schwann cells at some distance fromtheir point of secretion. Other forms may be released from intracells bysources via tissue injury and cell disruption. An example of a secretedGGF is the protein encoded by GGF2HBS5; this is the only GGF known whichhas been found to be directed to the exterior of the cell. Secretion isprobably mediated via an N-terminal hydrophobic sequence found only inregion E, which is the N-terminal domain contained within recombinantGGF2 encoded by GGF2HBS5.

Other GGF's appear to be non-secreted. These GGFs may be injury responseforms which are released as a consequence of tissue damage.

Other regions of the predicted protein structure of GGF2 (encoded byGGF2HBS5) and other proteins containing regions B and A exhibitsimilarities to the human basement membrane heparan sulfate proteoglycancore protein. The peptide ADSGEY, which is located next to the secondcysteine of the C2 immunoglobulin fold in these GGF's, occurs in nine oftwenty-two C-2 repeats found in that basal lamina protein. This evidencestrongly suggests that these proteins may associate with matrix proteinssuch as those associated with neurons and glia, and may suggest a methodfor sequestration of glial growth factors at target sites.

EXAMPLE 15

Purification of GGFs from Recombinant Cells

In order to obtain full length or portions of GGFs to assay forbiological activity, the proteins can be overproduced using cloned DNA.Several approaches can be used. A recombinant E. coli cell containingthe sequences described above can be constructed. Expression systemssuch as pNH8a or pHH16a (Stratagene, Inc.) can be used for this purposeby following manufacturers procedures. Alternatively, these sequencescan be inserted in a mammalian expression vector and an overproducingcell line can be constructed. As an example, for this purpose DNAencoding a GGF, clone GGF2BPP5 has been expressed in COS cells and canbe expressed in Chinese hamster ovary cells using the pMSXND expressionvector (Lee and Nathans, J. Biol. Chem. 263, 3521–3527, (1981)). Thisvector containing GGF DNA sequences can be transfected into host cellsusing established procedures.

Transient expression can be examined or G418-resistant clones can begrown in the presence of methotrexate to select for cells that amplifythe dhfr gene (contained on the pMSXND vector) and, in the process,co-amplify the adjacent GGF protein encoding sequence. Because CHO cellscan be maintained in a totally protein-free medium (Hamilton and Ham, InVitro 13, 537–547 (1977)), the desired protein can be purified from themedium. Western analysis using the antisera produced in Example 16 canbe used to detect the presence of the desired protein in the conditionedmedium of the overproducing cells.

The desired protein (rGGF2) was purified from the medium conditioned bytransiently expressing cos cells as follows. rGGF II was harvested fromthe conditioned medium and partially purified using Cation ExchangeChromatography (POROS-HS). The column was equilibrated with 33.3 mM MESpH 6.0. Conditioned media was loaded at flow rate of 10 ml/min. The peakcontaining Schwann cell proliferation activity and immunoreactive (usingthe polyclonal antisera was against a GGF2 peptide described above) waseluted with 50 mM Tris, 1M NaCl pH 8.0.

rhGGF2 is also expressed using a stable Chinese Ovary Hamster cell line.rGGF2 from the harvested conditioned media was partially purified usingCation Exchange Chromatograph (POROS-HS). The column was equilibratedwith PBS pH 7.4. Conditioned media was loaded at 10 ml/min. The peakcontaining the Schwann Cell Proliferative activity and immunoreactivity(using GGF2 polyclonal antisera) was eluted with 50 mM Hepes, 500 mMNaCl pH 8.0. An additional peak was observed at 50 mM Hepes, 1M NaCl pH8.0 with both proliferation as well as immunoreactivity (FIG. 45).

rhGGF2 can be further purified using Hydrophobic InteractionChromatography as a high resolution step; Cation exchange/Reserve phaseChromatography (if needed as second high resolution step); A viralinactivation step and a DNA removal step such as Anion exchangechromatography.

Schwann Cell Proliferation Activity of recombinant GGF2 peak eluted fromthe Cation Exchange column was determined as follows: Mitogenicresponses of the cultured Schwann cells were measured in the presence of5 M Forskolin using the peak eluted by 50 mM Tris 1 M NaCl pH 8.0. Thepeak was added at 20 1, 10 1 (1:10) 10 1 and (1:100) 10 1. Incorporationof ¹²⁵I-Uridine was determined and expressed as (CPM) following an 18–24hour exposure.

An immunoblot using polyclonal antibody raised against a peptide of GGF2was carried out as follows: 10 1 of different fractions were ran on4–12% gradient gels. The gels were transferred on to Nitrocellulosepaper, and the nitrocellulose blots were blocked with 5% BSA and probedwith GGF2-specific antibody (1:250 dilution). ¹²⁵I protein A (1:500dilution, Specific Activity=9.0/Ci/g) was used as the secondaryantibody. The immunoblots were exposed to Kodax X-Ray films for 6 hours.The peak fractions eluted with 1 M NaCl showed an immunoreactive band at69K.

GGF2 purification on cation exchange columns was performed as follows:CHO cell conditioned media expressing rGGFII was loaded on the cationexchange column at 10 ml/min. The column was equilibrated with PBS pH7.4. The elution was achieved with 50 mM Hepes 500 mM NaCl pH 8.0 and 50mM Hepes 1M NaCl pH 8.0 respectively. All fractions were analyzed usingthe Schwann cell proliferation assay (CPM) described herein. The proteinconcentration (mg/ml) was determined by the Bradford assay using BSA asthe standard.

A Western blot using 10 1 of each fraction was performed andimmunoreactivity and the Schwann cell activity were observed toco-migrate.

The protein may be assayed at various points in the procedure using aWestern blot assay. Alternatively, the Schwann cell mitogenic assaydescribed herein may be used to assay the expressed product of the fulllength clone or any biologically active portions thereof. The fulllength clone GGF2BPP5 has been expressed transiently in COS cells.Intracellular extracts of transfected COS cells show biological activitywhen assayed in the Schwann cell proliferation assay described inExample 8. In addition, the full length close encoding GGF2HBS5 has beenexpressed transiently in COS cells. In this case both cell extract andconditioned media show biological activity in the Schwann cellproliferation assay described in Example 8. Any member of the family ofsplicing variant complementary DNA's derived from the GGF gene(including the Heregulins) can be expressed in this manner and assayedin the Schwann cell proliferation assay by one skilled in the art.

Alternatively, recombinant material may be isolated from other variantsaccording to Wen et al. (Cell 69:559 (1992)) who expressed the splicingvariant Neu differentiation factor (NDF) in COS-7 cells. cDNA clonesinserted in the pJT-2 eukaryotic plasmid vector are under the control ofthe SV40 early promoter, and are 3′-flanked with the SV40 terminationand polyadenylation signals. COS-7 cells were transfected with the pJT-2plasmid DNA by electroporation as follows: 6×10⁶ cells (in 0.8 ml ofDMEM and 10% FEBS) were transferred to a 0.4 cm cuvette and mixed with20 μg of plasmid DNA in 10 μl of TE solution (10 mM Tris-HCl (pH 8.0), 1mM EDTA). Electroporation was performed at room temperature at 1600 Vand 25 μF using a Bio-Rad Gene Pulser apparatus with the pulsecontroller unit set at 200 ohms. The cells were then diluted into 20 mlof DMEM, 10% FBS and transferred into a T75 flask (Falcon). After 14 hr.of incubation at 37° C., the medium was replaced with DMEM, 1% FBS, andthe incubation continued for an additional 48 hr. Conditioned mediumcontaining recombinant protein which was harvested from the cellsdemonstrated biological activity in a cell line expressing the receptorfor this protein. This cell line (cultured human breast carcinoma cellline AU 565) was treated with recombinant material. The treated cellsexhibited a morphology change which is characteristic of the activationof the erbB2 receptor. Conditioned medium of this type also can betested in the Schwann cell proliferation assay.

EXAMPLE 16

Isolation of a Further Splicing Variant

Methods for updating other neuregulins descsribed in U.S. patentapplication Ser. No. 07/965,173, filed Oct. 23, 1992, incorporatedherein by reference, produced four closely related sequences (heregulinα, β1, β2, β3) which arise as a result of splicing variation. Peles etal. (Cell 69:205 (1992)), and Wen et al. (Cell 69:559 (1992)) haveisolated another splicing variant (from rat) using a similarpurification and cloning approach to that described in Examples 1–9 and11 involving a protein which binds to p185^(erbB2). The cDNA clone wasobtained as follows (via the purification and sequencing of ap185^(erbB2) binding protein from a transformed rat fibroblast cellline). A p185^(erbB2) binding protein was purified from conditionedmedium as follows. Pooled conditioned medium from three harvests of 500roller bottles (120 liters total) was cleared by filtration through 0.2μfilters and concentrated 31-fold with a Pelicon ultrafiltration systemusing membranes with a 20 kd molecular size cutoff. All the purificationsteps were performed by using a Pharmacia fast protein liquidchromatography system. The concentrated material was directly loaded ona column of heparin-Sepharose (150 ml, preequilibrated withphosphate-buffered saline (PBS)). The column was washed with PBScontaining 0.2 M NaCl until no absorbance at 280 nm wavelength could bedetected. Bound proteins were then eluted with a continuous gradient(250 ml) of NaCl (from 0.2 M to 1.0 M), and 5 ml fractions werecollected. Samples (0.01 ml of the collected fractions were used for thequantitative assay of the kinase stimulatory activity. Active fractionsfrom three column runs (total volume=360 ml) were pooled, concentratedto 25 ml by using a YM10 ultrafiltration membrane (Amicon, Danvers,Mass.), and ammonium sulfate was added to reach a concentration of 1.7M. After clearance by centrifugation (10,000×g, 15 min.), the pooledmaterial was loaded on a phenyl-Superose column (HR10/10, Pharmacia).The column was developed with a 45 ml gradient of (NH₄)₂SO₄ (from 1.7 Mto no salt) in 0.1 M Na₂PO₄ (pH 7.4), and 2 ml fractions were collectedand assayed (0.002 ml per sample) for kinase stimulation (as describedin Example 18). The major peak of activity was pooled and dialyzedagainst 50 mM sodium phosphate buffer (pH 7.3). A Mono-S cation-exchangecolumn (HR5/5, Pharmacia) was preequilibrated with 50 mM sodiumphosphate. After loading the active material (0.884 mg of protein; 35ml), the column was washed with the starting buffer and then developedat a rate of 1 ml/min. with a gradient of NaCl. The kinase stimulatoryactivity was recovered at 0.45–0.55 M salt and was spread over fourfractions of 2 ml each. These were pooled and loaded directly on a Cu⁺²chelating columns (1.6 ml, HR2/5 chelating Superose, Pharmacia). Most ofthe proteins adsorbed to the resin, but they gradually eluted with a 30ml linear gradient of ammonium chloride (0–1 M). The activity eluted ina single peak of protein at the range of 0.05 to 0.2 M NH₄Cl. Samplesfrom various steps of purification were analyzed by gel electrophoresisfollowed by silver staining using a kit from ICN (Costa Mesa, Calif.),and their protein contents were determined with a Coomassie blue dyebinding assay using a kit from Bio-Rad (Richmond, Calif.).

The p44 protein (10 μg) was reconstituted in 200 μl of 0.1 M ammoniumbicarbonate buffer (pH 7.8). Digestion was conducted withL-1-tosyl-amide 2-phenylethyl chloromethyl ketone-treated trypsin(Serva) at 37° C. for 18 hr. at an enzyme-to-substrate ratio of 1:10.The resulting peptide mixture was separated by reverse-phase HPLC andmonitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d.×15 cm,300 Å) and an HP 1090 liquid chromatographic system equipped with adiode-array detector and a workstation. The column was equilibrated with0.1% trifluoroacetic acid (mobile phase A), and elution was effectedwith a linear gradient from 0%–55% mobile phase B (90% acetonitrile in0.1% trifluoroacetic acid) over 70 min. The flow rate was 0.2 ml/min.and the column temperature was controlled at 25° C. One-third aliquotsof the peptide peaks collected manually from the HPLC system werecharacterized by N-terminal sequence analysis by Edman degradation. Thefraction eluted after 27.7 min. (T27.7) contained mixed amino acidsequences and was further rechromatographed after reduction as follows:A 70% aliquot of the peptide fraction was dried in vacuo andreconstituted in 100 μl of 0.2 M ammonium bicarbonate buffer (pH 7.8).DTT (final concentration 2 mM) was added to the solution, which was thenincubated at 37° C. for 30 min. The reduced peptide mixture was thenseparated by reverse-phase HPLC using a Vydac column (2.1 mm i.d.×15cm). Elution conditions and flow rat were identical to those describedabove. Amino acid sequence analysis of the peptide was performed with aModel 477 protein sequencer (Applied Biosystems, Inc., Foster City,Calif.) equipped with an on-line phenylthiohydantoin (PTH) amino acidanalyzer and a Model 900 data analysis system (Hunkapiller et al. (1986)In Methods of Protein Microcharacterization, J. E. Shively, ed.(Clifton, N.J.: Humana Press p. 223–247). The protein was loaded onto atrifluoroacetic acid-treated glass fiber disc precycled with polybreneand NaCl. The PTH-amino acid analysis was performed with a micro liquidchromatography system (Model 120) using dual syringe pumps andreverse-phase (C-18) narrow bore columns (Applied Biosystems, 2.1 mm×250mm).

RNA was isolated from Rat1-EJ cells by standard procedures (Maniatis etal., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.(1982) and poly (A)⁺ was selected using an mRNA Separator kit (ClontechLab, Inc., Palo Alto, Calif.). cDNA was synthesized with the Superscriptkit (from BRL Life Technologies, Inc., Bethesda, Md.).Column-fractionated double-strand cDNA was ligated into an Sal1- andNot1-digested pJT-2 plasmid vector, a derivative of the pCD-X vector(Okayama and Berg, Mol. Cell Biol. 3: 280 (1983)) and transformed intoDH10B E. coli cells by electroporation (Dower et al., Nucl. Acids Res.16: 6127 (1988)). Approximately 5×10⁵ primary transformants werescreened with two oligonucleotide probes that were derived from theprotein sequences of the N-terminus of NDF (residues 5–24) and the T40.4tryptic peptide (residues 7–12). Their respective sequences were asfollows (N indicates all 4 nt):

-   (1) 5′-ATA GGG AAG GGC GGG GGA AGG GTC NCC CTC NGC    -   A T    -   AGG GCC GGG CTT GCC TCT GGA GCC TCT-3′-   (2) 5′-TTT ACA CAT ATA TTC NCC-3′    -   C G G C-   (1: SEQ ID NO: 163; 2: SEQ ID NO: 164)    The synthetic oligonucleotides were end-labeled with [γ-³²P]ATP with    T4 polynucleotide kinase and used to screen replicate sets of    nitrocellulose filters. The hybridization solution contained 6×SSC,    50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 2×    Denhardt's solution, 50 μg/ml salmon sperm DNA, and 20% formamide    (for probe 1) or no formamide (for probe 2). The filters were washed    at either 50° C. with 0.5×SSC, 0.2% SDS, 2 mM EDTA (for probe 1) or    at 37° C. with 2×SSC, 0.2% SDS, 2 mM EDTA (for probe 2).    Autoradiography of the filters gave ten clones that hybridized with    both probes. These clones were purified by replating and probe    hybridization as described above. The cDNA clones were sequenced    using an Applied Biosystems 373A automated DNA sequencer and Applied    Biosystems Taq DyeDeoxy™ Terminator cycle sequencing kits following    the manufacture's instructions. In some instances, sequences were    obtained using [³⁵S]dATP (Amersham) and Sequenase™ kits from U.S.    Biochemicals following the manufacturer's instructions. Both strands    of the cDNA clone 44 were sequenced by using synthetic    oligonucleotides as primers. The sequence of the most 5′ 350 nt was    determined in seven independent cDNA clones. The resultant clone    demonstrated the pattern shown in FIG. 27 (NDF).

EXAMPLE 17

Purification and Assay of Other Proteins which Bind p185^(erbB2)Receptor

I. Purification of gp30 and p70

Lupu et al. (Science 249, 1552 (1990)) and Lippman and Lupu (patentapplication number PCT/US91/03443 (1990)), hereby incorporated byreference, have purified a protein from conditioned media of a humanbreast cancer cell line MDA-MB-231.

Lupu et al. (Proc. Natl. Acad. Sci. 89, 2287 (1992)) purified anotherprotein which binds to the p185^(erbB2) receptor. This particularprotein, p75, was purified from conditioned medium used for the growthof SKBr-3 (a human breast cancer cell line) propagated in improvedEagle's medium (IMEM: GIBCO) supplemented with 10% fetal bovine serum(GIBCO).

II. Other p185^(erbB2) Ligands

Peles et al. (Cell 69, 205 (1992)) have also purified a p185erB2stimulating ligand from rat cells. Holmes et al. (Science 256, 1205(1992)) have purified Heregulin α from human cells which binds andstimulates p185erB2 (see Example 5). Tarakovsky et al. Oncogene 6:218(1991) have demonstrated binding of a 25 kD polypeptide isolated fromactivated macrophages to the Neu receptor, a p185^(erbB2) homolog,herein incorporated by reference.

III. NDF Isolation

Yarden and Peles (Biochemistry 30, 3543 (1991)) have identified a 35kilodalton glycoprotein which will stimulate the p185erB2 receptor.

In other publications, Davis et al. (Biochem. Biophys. Res. Commun. 179,1536 (1991), Proc. Natl. Acad. Sci. 88, 8582 (1991) and Greene et al.,PCT patent application PCT/US91/02331 (1990)) describe the purificationof a protein from conditioned medium of a human T-cell (ATL-2) cellline.

Huang et al. (1992, J. Biol. Chem. 257:11508–11512), hereby incorporatedby reference, have isolated an additional neu/erb B2 ligand growthfactor from bovine kidney. The 25 kD polypeptide factor was isolated bya procedure of column fractionation, followed by sequential columnchromatography on DEAE/cellulose (DE52), Sulfadex (sulfated SephadexG-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquidchromatography). The factor, NEL-GF, stimulates tyrosine-specificautophosphorylation of the neu/erb B2 gene product.

IV. Purification of Acetylcholine Receptor Inducing Activity (ARIA)

ARIA, a 42 kD protein which stimulates acetylcholine receptor synthesis,has been isolated in the laboratory of Gerald Fischbach (Falls et al.,(1993) Cell 72:801–815). ARIA induces tyrosine phosphorylation of a 185Kda muscle transmembrane protein which resembles p185^(erbB2), andstimulates acetylcholine receptor synthesis in cultured embryonicmyotubes. ARIA is most likely a member of the GGF/erbB2 ligand group ofproteins, and this is potentially useful in the glial cell mitogenesisstimulation and other applications of, e.g., GGF2 described herein.

EXAMPLE 18

Protein Tyrosine Phosphorylation Mediated by GGF

Rat Schwann cells, following treatment with sufficient levels of GlialGrowth Factor to induce proliferation, show stimulation of proteintyrosine phosphorylation. Varying amounts of partially purified GGF wereapplied to a primary culture of rat Schwann cells according to theprocedure outlined in Example 9. Schwann cells were grown in DMEM/10%fetal calf serum/5 μM forskolin/0.5 μg per mL GGF-CM (0.5 mL per well)in poly D-lysine coated 24 well plates. When confluent, the cells werefed with DMEM/10% fetal calf serum at 0.5 mL per well and left in theincubator overnight to quiesce. The following day, the cells were fedwith 0.2 mL of DMEM/10% fetal calf serum and left in the incubator for 1hour. Test samples were then added directly to the medium at differentconcentrations and for different lengths of time as required. The cellswere then lysed in boiling lysis buffer (sodium phosphate, 5 mM, pH 6.8;SDS, 2%, β-mercapteothanol, 5%; dithiothreitol, 0.1M; glycerol, 10%;Bromophenol Blue, 0.4%; sodium vanadate, 10 mM), incubated in a boilingwater bath for 10 minutes and then either analyzed directly or frozen at−70° C. Samples were analyzed by running on 7.5% SDS-PAGE gels and thenelectroblotting onto nitrocellulose using standard procedures asdescribed by Towbin et al. (1979) Proc. Natl. Acad. Sci. USA76:4350–4354. The blotted nitrocellulose was probed withantiphosphotyrosine antibodies using standard methods as described inKamps and Selton (1988) Oncogene 2:305–315. The probed blots wereexposed to autoradiography film overnight and developed using a standardlaboratory processor. Densitometric measurements were carried out usingan Ultrascan XL enhanced laser densitometer (LKB). Molecular weightassignments were made relative to prestained high molecular weightstandards (Sigma). The dose responses of protein phosphorylation andSchwann cell proliferation are very similar (FIG. 33). The molecularweight of the phosphorylated band is very close to the molecular weightof p185^(erbB2). Similar results were obtained when Schwann cells weretreated with conditioned media prepared from COS cells translates withthe GGF2HBS5 clone. These results correlate well with the expectedbinging and activation of p185erB2 by the GGFS.

This experiment has been repeated with recombinant GGF2. Conditionedmedium derived from a CHO cell line stably transformed with the GGF2clone (GGF2HBS5) stimulates protein tyrosine phosphorylation using theassay described above. Mock transfected CHO cells fail to stimulate thisactivity.

EXAMPLE 19

N-glycosylation of GGF

The protein sequence predicted from the cDNA sequence of GGF-IIcandidate clones GGF2BPP1, 2 and 3 contains a number of consensusN-glycosylation motifs. A gap in the GGFII02 peptide sequence coincideswith the asparagine residue in one of these motifs, indicating thatcarbohydrate is probably bound at this site.

N-glycosylation of the GGFs was studied by observing mobility changes onSDS-PAGE after incubation with N-glycanase, an enzyme that cleaves thecovalent linkages between carbohydrate and aspargine residues inproteins.

N-Glycanase treatment of GGF-II yielded a major band of MW 40–42 kDa anda minor band at 45–48 kDa. Activity single active deglycosylated speciesat ca 45–50 kDa.

Activity elution experiments with GGF-I also demonstrate an increase inelectrophoretic mobility when treated with N-Glycanase, giving an activespecies of MW 26–28 kDa. Silver staining confirmed that there is amobility shift, although no N-deglycosylated band could be assignedbecause of background staining in the sample used.

Further embodiments are within the following claims.

1. A method of increasing myotube formation, myotube survival, musclecell mitogenesis, or muscle cell survival in a mammal in need thereof,said method comprising administering to said mammal in an amounteffective for increasing said myotube formation, myotube survival,muscle cell mitogenesis, or muscle cell survival, a polypeptidecomprising an amino acid sequence of SEQ ID NO:
 324. 2. The method ofclaim 1, wherein said mammal has a condition which involves muscledamage.
 3. The method of claim 1, wherein said increasing results indecreased atrophy of said muscle cell.
 4. The method of claim 1, whereinsaid increasing results in increased muscle fibers present in saidmammal.
 5. The method of claim 1, wherein said increasing is increasingthe survival of said muscle cell.
 6. The method of claim 1, wherein saidincreasing results in increased muscle growth in said mammal.
 7. Themethod of claim 1, wherein said increasing results in increased muscleregeneration in said mammal.
 8. The method of claim 1, wherein saidincreasing is increasing the mitogenesis of said muscle cell.
 9. Themethod of claim 1, wherein said increasing further results in increasedacetylcholine receptor systhesis.
 10. The method of claim 1, whereinsaid mammal is a patient lacking a neurotrophic factor.
 11. The methodof claim 1, wherein said muscle cell is a myoblast.
 12. The method ofclaim 1, wherein said muscle cell is a satellite cell.
 13. The method ofclaim 1, wherein said muscle cell is in skeletal muscle.
 14. The methodof claim 1, wherein said muscle cell is in cardiac muscle.
 15. Themethod of claim 1, wherein said muscle cell is in smooth muscle.
 16. Themethod of claim 1, wherein said mammal is a patient with a skeletalmuscle disease.
 17. The method of claim 16, wherein said skeletal muscledisease is a myopathy.
 18. The method of claim 16, wherein said skeletalmuscle disease is a dystrophy.
 19. The method of claim 18, wherein saiddystrophy is Duchenne's muscular dystrophy.
 20. The method of claim 18,wherein said dystrophy is Becker's muscular dystrophy.
 21. The method ofclaim 16, wherein said skeletal muscle disease is a result of a neuralcondition.
 22. The method of claim 16, wherein said skeletal muscledisease is a traumatic injury.
 23. The method of claim 21, wherein saidcondition is a nerve injury.
 24. The method of claim 21, wherein saidcondition is a neuropathy.
 25. The method of claim 1, wherein saidmammal is a patient with a cardiac muscle disorder.
 26. The method ofclaim 25, wherein said disorder is cardiomyopathy.
 27. The method ofclaim 25, wherein said disorder is ischemic damage.
 28. The method ofclaim 25, wherein said disorder is a degenerative congenital cardiacdisease.
 29. The method of claim 25, wherein said disorder is cardiactrauma.
 30. The method of claim 1, wherein said mammal is a patient witha smooth muscle disorder.
 31. The method of claim 30, wherein saiddisorder is atherosclerosis and said increasing is increasing thedifferentiation of said muscle cell.
 32. The method of claim 30, whereinsaid disorder is a vascular lesion.
 33. The method of claim 30, whereinsaid disorder is a degenerative congenital vascular disease.
 34. Themethod of claim 1, wherein said muscle cell has insufficient functionalacetylcholine receptors.
 35. The method of claim 34, wherein said musclecell lacking sufficient acetylcholine receptors is in a patient withmyasthenia gravis.
 36. The method of claim 30, wherein said disorder isarterial sclerosis.